Increasing salt tolerance in plants by overexpression of a vacuolar Na+/H+ transporter[s]

ABSTRACT

The invention is isolated nucleic acid molecules encoding Na + /H +  exchanger polypeptides with 95% identity to a Na + /H +  exchanger polypeptide from  Arabidopsis thaliana  for extrusion of monovalent cations from the cytosol of cells to provide the cell with increased salt tolerance. Crop species transformed with the nucleic acid molecule are capable of surviving in soil with high salt levels that would normally inhibit growth of the crop species.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 09/271,584 filed Mar. 18, 1999, which claimspriority from U.S. application Ser. No. 60/078,474 filed Mar. 18, 1998and U.S. application Ser. No. 60/116,111 filed Jan. 15, 1999, all ofwhich are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Environmental stress due to salinity is one of the most serious factorslimiting the productivity of agricultural crops, which are predominantlysensitive to the presence of high concentrations of salts in the soil.Large terrestrial areas of the world are affected by levels of saltinimical to plant growth. It is estimated that 35-45% of the 279 millionhectares of land under irrigation is presently affected by salinity.This is exclusive of the regions classified as arid and desert lands,(which comprises 25% of the total land of our planet). Salinity has beenan important factor in human history and in the life spans ofagricultural systems. Salt impinging on agricultural soils has createdinstability and has frequently destroyed ancient and recent agrariansocieties. The Sumerian culture faded as a power in the ancient worlddue to salt accumulation in the valleys of the Euphrates and Tigrisrivers. Large areas of the Indian subcontinent have been renderedunproductive through salt accumulation and poor irrigation practices. Inthis century, other areas, including vast regions of Australia, Europe,southwest USA, the Canadian prairies and others have seen considerabledeclines in crop productivity.

Although there is engineering technology available to combat thisproblem, though drainage and supply of high quality water, thesemeasures are extremely costly. In most of the cases, due to theincreased need for extensive agriculture, neither improved irrigationefficiency nor the installation of drainage systems is applicable.Moreover, in the arid and semi-arid regions of the world waterevaporation exceeds precipitation. These soils are inherently high insalt and require vast amounts of irrigation to become productive. Sinceirrigation water contains dissolved salts and minerals, an applicationof water is also an application of salt that compounds the salinityproblem.

Increasing emphasis is being given to modify plants to fit therestrictive growing conditions imposed by salinity. If economicallyimportant crops could be manipulated and made salt resistant, this landcould be farmed resulting in greater sales of seed and greater yield ofuseful crops. Conventional breeding for salt tolerance has beenattempted for a long time. These breeding practices have been basedmainly on the following strategies: a) the use of wide crosses betweencrop plants and their more salt-tolerant wild relatives [1], b)screening and selecting for variation within a particular phenotype [2],c) designing new phenotypes through recurrent selection [3]. The lack ofsuccess in generating tolerant varieties (given the low number ofvarieties released and their limited salt tolerance) [4] would suggestthat conventional breeding practices are not enough and that in order tosucceed a breeding program should include the engineering of transgeniccrops [5].

Several biochemical pathways associated with stress tolerance have beencharacterized in different plants and a few of the genes involved inthese processes have been identified and in some cases the possible roleof proteins has been investigated in transgenic/overexpressionexperiments. Several compatible solutes have been proposed to play arole in osmoregulation under stress. Such compatible solutes, includingcarbohydrates [6], amino acids [7] and quaternary N-compounds [8] havebeen shown to increase osmoregulation under stress. Also, proteins thatare normally expressed during seed maturation (LEAs, Late EmbriogenesisAbundant proteins) have been suggested to play a role in water retentionand in the protection of other proteins during stress. Theoverexpression of LEA in rice provided a moderate benefit to the plantsduring water stress [9,10]. A single gene (sod2) coding for a Na⁺/H⁺antiport has been shown to confer sodium tolerance in fission yeast[11,12], although the role of this plasma membrane-bound protein appearsto be only limited to yeast. One of the main disadvantages of using thisgene for transformation of plants is associated with the typicalproblems encountered in heterologous gene expression, i.e. incorrectfolding of the gene product, targeting of the protein to the targetmembrane and regulation of the protein function.

Plants that tolerate and grow in saline environments have highintracellular salt levels. A major component of the osmotic adjustmentin these cells is accomplished by ion uptake. The utilization ofinorganic ions for osmotic adjustment suggests that salt-tolerant plantsmust be able to tolerate high levels of salts within their cells.However, enzymes extracted from these plants show high sensitivity tosalt. The sensitivity of the cytosolic enzymes to salt would suggestthat the maintenance of low cytosolic sodium concentration, either bycompartmentation in cell organelles or by exclusion through the plasmamembrane, must be necessary if the enzymes in the cell are to beprotected from the inimical effects of salt.

Plant cells are structurally well suited to the compartmentation of ions. Large membrane-bound vacuoles are the site for a considerable amountof sequestration of ions and other osmotically active substances. Acomparison of ion distribution in cells and tissues of various plantspecies indicates that a primary characteristic of salt tolerant plantsis their ability to exclude sodium out of the cell and to take up sodiumand to sequester it in the cell vacuoles. Transport mechanisms couldactively move ions into the vacuole, removing the potentially harmfulions from the cytosol. These ions, in turn, could act as an osmoticumwithin the vacuole, which would then be responsible for maintainingwater flow into the cell. Thus, at the cellular level both specifictransport systems for sodium accumulation in the vacuole and sodiumextrusion out of the cell are correlated with salt tolerance.

SUMMARY OF THE INVENTION

We have isolated the first such system of intracellular salt management.We identified the presence of a functional vacuolar Na⁺/H⁺ antiport inthe vacuolar membrane of higher plants [13,14,15,16,17,18].

We have demonstrated the Na⁺/H⁺ antiport function in isolated tonoplastmembranes and in intact vesicles and we showed that the activity ofantiport molecules was salt dependent. Neither a protein sequence nor agene encoding the antiport were identified in previously published work.We have now identified nucleic acid molecules coding for plant Na⁺/H⁺antiports, the nucleic acid molecules and polypeptides produced by thenucleic acid molecules being the subject of the present invention. Thesepolypeptides are useful for the extrusion of sodium ions from thecytosol, either through the accumulation of sodium ions into thevacuoles or into the extracellular space, thus providing the mostimportant trait for salt tolerance in plants. These nucleic acidmolecules, preferably genes, are useful for the engineering of salttolerant plants by transformation of salt-sensitive crops overexpressingone or more of these nucleic acid molecules under the control ofconstitutively active promoters or under the control ofconditionally-induced promoters. Agrobacterium tumefaciens-mediatedtransformation or particle-bombardment-mediated transformation areuseful for depending upon the plant species.

The invention includes an isolated nucleic acid molecule encoding a PNHXtransporter polypeptide, or a fragment of a polypeptide having Na⁺/H⁺transporter activity and capable of increasing salt tolerance in a cell.In particular, the present invention is directed to plant Na⁺/H⁺transporters having the nucleotide sequences depicted in SEQ ID NO: 1, 3and 5. The present invention is further directed to plant Na⁺/H⁺transporters having the protein sequences depicted in SEQ ID NO: 2, 4and 6. The present invention is further directed to plant Na⁺/H⁺transporters having nucleotide sequences encoding the protein sequencesdepicted in SEQ ID NO: 2, 4 and 6. The present invention is furtherdirected to transgenic plants expressing recombinant DNA sequencesencoding the protein sequences depicted in SEQ ID NO: 2, 4 and 6.

The invention also relates to an isolated nucleic acid molecule encodinga THX transporter polypeptide, PNHX transporter polypeptide, or afragment of a polypeptide having Na⁺/H⁺ transporter activity and capableof increasing salt tolerance in a cell, comprising a nucleic acidmolecule selected from the group consisting of:

-   -   (a) a nucleic acid molecule that hybridizes to all or part of a        nucleic molecule in FIG. 1 or a complement thereof under        moderate or high stringency hybridization conditions, wherein        the nucleic acid molecule encodes a TNHX transporter        polypeptide, a PNHX transporter polypeptide or a polypeptide        having Na⁺/H⁺ transporter activity and capable of increasing        salt tolerance in a cell;    -   (b) a nucleic acid molecule degenerate with respect to (a),        wherein the nucleic molecule encodes a TNHx transporter        polypeptide, a PNHX transporter polypeptide or a polypeptide        having Na⁺/H⁺ transporter activity and capable of increasing        salt tolerance in a cell.

The hybridization conditions preferably comprise moderate (also calledintermediate) or high stringency conditions selected from the conditionsin Table 4.

The invention also includes an isolated nucleic acid molecule encoding aTHX transporter polypeptide or a PNHX transporter polypeptide, or afragment of a polypeptide having Na⁺/H⁺ transporter activity and capableof increasing salt tolerances in a cell, comprising a nucleic acidmolecule selected from the group consisting of:

-   -   (a) the nucleic acid molecule of the coding strand shown in SEQ        ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, or a complement thereof;    -   (b) a nucleic acid molecule encoding the same amino acid        sequence as a nucleotide sequence of (a); and    -   (c) a nucleic acid molecule having at least 17% identity with        the nucleotide sequence of (a) and which encodes a THX        transporter polypeptide or the PNHX transporter polypeptide or a        polypeptide having Na⁺/H⁺ transporter activity.

The THX transporter polypeptide or the PNHX transporter polypeptidepreferably comprises an AtNHX transporter polypeptide. An AtNHXtransporter polypeptide is a polypeptide isolated from Arabidopsisthaliana having Na⁺/H⁺ transporter activity and capable of increasingsalt tolerance in a cell. The nucleic acid molecule may comprise all orpart of a nucleotide sequence in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5(or the coding region thereof).

The invention also includes an AtNHX nucleic acid molecule isolated fromArabidopsis thaliana, or a fragment thereof encoding a transporterpolypeptide having Na⁺/H⁺ transporter activity and capable of increasingsalt tolerance in a cell.

Another aspect of the invention relates to a recombinant nucleic acidmolecule comprising a nucleic acid molecule and a constitutive promotersequence or an inducible promoter sequence, operatively linked so thatthe promoter enhances transcription of the nucleic acid molecule in ahost cell.

The nucleic acid molecule preferably comprises genomic DNA, cDNA or RNA.In another aspect, the nucleic acid molecule is chemically synthesized.The nucleic acid molecule is preferably isolated from Arabidopsisthaliana.

The nucleic acid molecule preferably encodes a TNHX transporterpolypeptide or PNHX transporter polypeptide that is capable of extrudingmonovalent cations out of the cytosol of a cell to provide the cell withincreased salt tolerance, wherein the monovalent cations are selectedfrom at least one of the group consisting of sodium, lithium andpotassium. The cell preferably comprises a plant cell. The monovalentcations are preferably extruded into a vacuole or into the extracellularspace.

The invention also includes an isolated nucleic acid molecule comprisinga nucleic acid molecule selected from the group consisting of 8 to 10nucleotides of the nucleic acid molecules described above, 11 to 25nucleotides of the nucleic acid molecules described above, and 26 to 50nucleotides of the nucleic acid molecules described above.

Another aspect of the invention relates to a vector comprising a nucleicacid molecule of the invention. The vector preferably comprises apromoter selected from the group consisting of a super promoter, a 35Spromoter of cauliflower mosaic virus, a drought-inducible promoter, anABA-inducible promoter, a heat shock-inducible promoter, asalt-inducible promoter, a copper-inducible promoter, asteroid-inducible promoter and a tissue-specific promoter.

The invention also includes a host cell comprising a recombinant nucleicacid molecule of the invention, or progeny of the host cell.

The host cell is preferably selected from the group consisting of afungal cell, a yeast cell, a bacterial cell, a microorganism cell and aplant cell. The plant, a plant part, a seed, a plant cell or progenythereof preferably comprises the recombinant nucleic acid molecule ofthe invention. The plant part preferably comprises all or part of aleaf, a flower a stem, a root or a tuber. The plant, plant part, seed orplant cell is preferably of a species selected from the group consistingof potato, tomato, brassica, cotton, sunflower, strawberries, spinach,lettuce, rice, soybean, corn, wheat, rye, barley, atriplex, sorgum,alfalfa, salicornia and the plant species or types in the specification.

The plant, plant part, seed or plant cell preferably comprises a dicotplant or a monocot plant.

The invention also relates to a method for producing a recombinant hostcell capable of expressing the nucleic acid molecule of the invention,the method comprising introducing into the host cell a vector of theinvention. The invention also includes a method of producing agenetically transformed plant which expresses TNHX or PNHX transporterpolypeptide, comprising regenerating a genetically transformed plantfrom a plant cell, seed or plant part of the invention. In one method,the genome of the host cell also includes a functional TNHX or PNHXgene. In another method, the genome of the host cell does not include afunctional TNHX or PNHX gene. The invention also includes a transgenicplant produced according to a method of the invention.

Another aspect of the invention relates to a method for expressing aTNHX or PNHX transporter polypeptide in the host cell of the invention,a the plant, plant part, seed or plant cell of the invention, the methodcomprising culturing the host cell under conditions suitable for geneexpression. A method for producing a transgenic plant that expresseselevated levels of PNHX transporter polypeptide relative to anon-transgenic plant, comprising transforming a plant with the vector ofthe invention. The invention also relates to an isolated polypeptideencoded by and/or produced from a nucleic acid molecule of theinvention, or the vector of the invention.

The invention also relates to an isolated PNHX transporter polypeptideor a fragment thereof having Na⁺/H⁺ transporter activity and capable ofincreasing salt tolerance in a cell. The polypeptide of the inventionpreferably comprises an AtNHX transporter polypeptide. The polypeptideof the invention preferably comprises all or part of an amino acidsequence in SEQ ID NOS: 2, 4 or 6 (FIG. 1). The invention also includesa polypeptide fragment of the AtNHX transporter polypeptide of theinvention, or a peptide mimetic of the AtNHX transporter polypeptide,having Na⁺/H⁺ transporter activity and capable of increasing salttolerance in a cell. The polypeptide fragment of the invention,preferably consists of at least 20 amino acids, which fragment hasNa⁺/H⁺ transporter activity and is capable of increasing salt tolerancein a cell. The fragment or peptide mimetic of the invention ispreferably capable of being bound by an antibody to a polypeptide of theinvention. In one embodiment, the polypeptide of the invention isrecombinantly produced.

The invention also includes an isolated and purified transporterpolypeptide comprising the amino acid sequence of a TNHX transporterpolypeptide or a PNHX transporter polypeptide, wherein the transporterpolypeptide is encoded by a nucleic acid molecule that hybridizes undermoderate or stringent conditions to a nucleic acid molecule in SEQ IDNOS: 1, 3 or 5 (FIG. 1), a degenerate form thereof or a complement. Theinvention also includes a polypeptide comprising a sequence havinggreater than 28% sequence identity to a polypeptide of the invention(preferably a polypeptide in FIG. 1, such as SEQ ID NOS: 2, 4 or 6).

The polypeptide of the invention, preferably comprises a Na⁺/H⁺transporter polypeptide. The polypeptide is preferably isolated fromArabidopsis thaliana.

The invention also includes an isolated nucleic acid molecule encodingpolypeptide of the invention (preferably a polypeptide in FIG. 1: SEQ IDNOS: 2, 4, or 6).

Another aspect of the invention relates to an antibody directed againsta polypeptide of the invention. The antibody of the invention,preferably comprises a monoclonal antibody or a polyclonal antibody.

The invention relates to a method of producing a genetically transformedplant which expresses or overexpresses a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide having Na⁺/H⁺ transporteractivity and capable of increasing salt tolerance in a cell and whereinthe plant has increased salt tolerance, comprising:

-   -   a) cloning or synthesizing a TNHX nucleic acid molecule, a PNHX        nucleic acid molecule or a nucleic acid molecule which codes for        a Na⁺/H⁺ transporter polypeptide, wherein the polypeptide is        capable of providing salt tolerance to a plant;    -   b) inserting the nucleic acid molecule in a vector so that the        nucleic acid molecule is operably linked to a promoter;    -   c) inserting the vector into a plant cell or plant seed;    -   d) regenerating the plant from the plant cell or plant seed,        wherein salt tolerance in the plant is increased compared to a        wild type plant.

The invention includes a transgenic plant produced according to a methodof the invention.

The nucleic acid molecules have several uses which will be discussed inmore detail below. The nucleic acid molecules and the polypeptides areused in a method for protecting a plant from the adverse affects of asaline environment by incorporating a nucleic acid molecule for salttolerance and/or the polypeptide of the invention into a plant. Thenucleic acid molecules of the invention are also useful for theidentification of homologous nucleic acid molecules from plant species,preferably salt tolerant species and genetically engineering salttolerant plants of agricultural and commercial interest.

The invention relates to isolated nucleic acid molecules encoding apolypeptide for extrusion of sodium ions from the cytosol of a cell toprovide the cell with salt tolerance. The nucleic acid moleculespreferably comprise the nucleotide sequence in FIGS. 1(a) to 1(c). Thenucleic acid molecules may be DNA or RNA. The nucleic acid molecules maybe used to transform a cell selected from the group consisting of aplant cell, a yeast cell and a bacterial cell. The sodium ions areextruded into a vacuole or out of the cell. The nucleic acid moleculesencode a Na⁺/H⁺ exchanger polypeptide.

In a preferred embodiment, the nucleic acid molecules are isolated fromArabidopsis thaliana.

The invention includes an isolated nucleic acid molecule, comprising theDNA sequence in FIGS. 1(a), (b) (c). The invention also relates to anisolated nucleic acid molecule, comprising a sequence having greaterthan 17% homology to the sequences of the invention described in thepreceding paragraphs.

In an alternate embodiment, the nucleic acid molecule consists of asequence selected from the group consisting of 8 to 10 nucleotides ofthe nucleic acid molecules of the invention, 11 to 25 nucleotides of thenucleic acid molecule and 26 to 50 nucleotides of the nucleic acidmolecules. These nucleic acid molecules hybridize to nucleic acidmolecules described in the preceding paragraphs.

The nucleic acid molecule of the invention may have a sense or anantisense sequence.

In an alternate embodiment, the invention is an expression vectorcomprising a nucleic acid molecule of the invention. The expressionvector preferably consists of a promoter selected from the groupconsisting of a super promoter, a 35S promoter of cauliflower mosaicvirus, a drought-inducible promoter, an ABA-inducible promoter, a heatshock-inducible promoter, a salt-inducible promoter, a copper-induciblepromoter, a steroid inducible promoter and a tissue-specific promoter.

The invention is a polypeptide produced from the nucleic acid moleculesof the invention. The invention is also a polypeptide produced from theexpression vector. The polypeptide is used for extrusion of sodium ionsfrom the cytosol of a cell to provide the cell with salt tolerance.

In a preferred embodiment, the polypeptide has the amino acid sequencein FIGS. 1(a)-(e). The polypeptides may be homologous to the polypeptidein FIGS. 1(a)-(e). In an alternate embodiment, the polypeptides comprisea sequence having greater than 28% homology to the polypeptide in FIGS.1(a)-(e). The polypeptides are Na⁺/H⁺ exchanger polypeptides.

The polypeptides are preferably isolated from Arabidopsis thaliana.

The invention also includes a monoclonal antibody or polyclonal antibodydirected against a polypeptide of the invention.

Another embodiment of the invention includes a transformed microorganismcomprising an isolated nucleic acid molecule of the invention. Theinvention also includes a transformed microorganism including anexpression vector.

The invention includes a plant cell transformed with a nucleic acidmolecule of the invention. The invention also includes a yeast celltransformed with the nucleic acid molecule of the invention. In anotherembodiment, the invention is a plant, plant part or seed, generated froma plant cell transformed with a nucleic acid molecule of the invention.The invention also relates to a plant, plant part, seed or plant celltransfected with a nucleic acid molecule of the invention. The plant,plant part, seed or plant cell is preferably selected from a speciesselected from the group consisting of potato, tomato, brassica, cotton,sunflower, strawberries, spinach, lettuce, rice, soybean, corn, wheat,rye, barley, atriplex, sorgum, alfalfa and salicornia and other plantsdescribed herein.

The invention also includes a method for producing a polypeptide of theinvention by culturing a plant, plant part, seed or plant cell of theinvention and recovering the expressed polypeptide from the culture.

The invention includes an isolated nucleic acid molecule encoding apolypeptide capable of extruding monovalent cations from the cytosol ofa cell to provide the cell with increased salt tolerance. The nucleicacid molecule preferably includes the nucleotide sequences in FIG. 1.The nucleic acid molecule is preferably DNA or RNA. The cell ispreferably a plant cell, a yeast cell or a bacterial cell. Themonovalent cations are preferably sodium, lithium or potassium. Themonovalent cations are preferably extruded into a vacuole or out of thecell. The nucleic acid molecules preferably encode a Na⁺/H⁺ exchangerpolypeptide. The nucleic acid molecule is preferably isolated fromArabidopsis thaliana.

The invention also includes an isolated nucleic acid molecule, includinga sequence having greater than 17% homology to a sequence referred to inthe preceding paragraph.

The invention also includes a nucleic acid molecule of 8 to 10nucleotides, 11 to 25 or 26 to 50 nucleotides of a nucleic acid moleculeof the invention.

The invention also includes a nucleic acid molecule which nucleic acidmolecule hybridizes a nucleic acid molecule of the invention. Thenucleic acid molecule comprises a sense or an antisense sequence.

The invention also includes an expression vector including a nucleicacid molecule of the invention. The expression vector preferablycomprises a promoter selected from the group consisting of a superpromoter, a 35S promoter of cauliflower mosaic virus, adrought-inducible promoter, an ABA-inducible promoter, a heatshock-inducible promoter, a salt-inducible promoter, a copper-induciblepromoter, a steroid-inducible promoter and a tissue-specific promoter.

The invention also includes a polypeptide produced from a nucleic acidmolecule or expression vector of the invention. The invention alsoincludes a polypeptide for extrusion of monovalent cations ions from thecytosol of a cell to provide the cell with salt tolerance. The inventionalso includes a polypeptide including the amino acid sequence in FIG. 1or a polypeptide homologous to one of these sequences. The inventionalso includes a polypeptide including a sequence having greater than 28%homology to one of these polypeptides. The polypeptide is preferably aNa⁺/H⁺ exchanger polypeptide isolated from Arabidopsis thaliana. Theinvention also includes a peptide including at least 5 amino acids or 41to 75 amino acids of the polypeptide of the invention. The inventionalso includes nucleic acid molecules these polypeptides.

The invention includes a polypeptide for extrusion of monovalent cationsions from the cytosol of a cell to provide the cell with salt tolerance,including, but not necessarily having, an amiloride binding domain.

Another aspect of the invention relates to a monoclonal or polyclonalantibody directed against a polypeptide of the invention.

Another variation includes a transformed microorganism including anisolated nucleic acid molecule of the invention. The transformedmicroorganism preferably includes an expression vector of the invention.

As shown in Table 2 below, many nucleic acid molecules identified inArabidopsis thaliana have striking DNA sequence similarity to nucleicacid molecules encoding the homologous polypeptide in other plantspecies. Using the techniques described in this application and othersknown in the art, it will be apparent that the nucleic acid moleculeencoding the homologous Na⁺/H⁺ exchanger in other plant speciesincluding, but not limited to plants of agricultural and commercialinterest, will have DNA sequence identity (homology) at leastabout >17%, >20%, >25%, >35% to a DNA sequence shown in FIG. 1 or 5 (ora partial sequence thereof). Some plants species may have DNA with asequence identity (homology) at least about: >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to a DNAsequence in FIG. 1 or 5 (or a partial sequence thereof). The inventionalso includes modified nucleic acid molecules from plants other thanArabidopsis thaliana which have sequence identity at leastabout: >17%, >20%, >25%, >35%, >50%, >60%, >70% , >80% or >90% morepreferably at least about >95%, >99% or >99.5%, to an AtNHX sequence inFIG. 1 or 5 (or a partial sequence thereof). Modified nucleic acidmolecules are discussed below. Preferably about 1, 2, 3, 4, 5, 6 to 10,10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides or aminoacids are modified. Sequence identity is most preferably calculated asthe number of identical amino acid residues expressed as a percentage ofthe length of the shorter of the two sequences in a pairwise alignment.The pairwise alignment is constructed preferably using the Clustal Wprogram preferably using the following parameter settings: fixed gappenalty=10, floating gap penalty=10, protein weight matrix=BLOSUM62. Forexample, if a nucleotide sequence (called “Sequence A”) has 90% identityto a portion of the nucleotide sequence in FIG. 1(a), then Sequence Awill be identical to the referenced portion of the nucleotide sequencein FIG. 8, except that Sequence A may include up to 10 point mutations,such as substitutions with other nucleotides, per each 100 nucleotidesof the referenced portion of the nucleotide sequence in FIG. 8.Polypeptides having sequence identity may be similarly identified.

The invention includes a nucleic acid molecule that encodes all or partof a polypeptide capable of extruding monovalent cations from thecytosol of a cell to provide the cell with salt tolerance, wherein thesequence hybridizes to the nucleic acid molecule of all or part of SEQID NO: 1 or SEQ ID NO:3, SEQ ID NO:5 under low, medium and highstringency conditions.

The invention includes an isolated nucleic acid molecule encoding apolypeptide capable of extruding monovalent cations from the cytosol ofa cell to provide the cell with salt tolerance, including at least oneof the nucleic acid molecules in FIG. 1. The molecule is preferably DNAor RNA. The cell is preferably selected from the group consisting of aplant cell, a yeast cell and a bacterial cell and encodes a Na⁺/H⁺exchanger polypeptide isolated from Arabidopsis thaliana.

It will be clear to one skilled in the art that the sequences in FIG. 1are useful in isolating other salt tolerant nucleic acid molecules (forexample probes may be made from the sequences in FIG. 1, preparingtransgenic plants and performing many of the other methods of theinvention that are described with respect to sequences in FIG. 1.Variants and modifications of FIG. 1 sequences are also included withinthe invention as are methods using varied or modified sequences (thesame preferred percentages of identity and sequence described withrespect to FIG. 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in relation to thedrawings in which:

FIG. 1(a) Shows the nucleic acid molecule that is SEQ ID NO:1 and thepolypeptide that is SEQ ID NO:2. In a preferred embodiment, the figureshows isolated AtNHX2 cDNA encoding a Na⁺/H⁺ exchanger from Arabidopsisthaliana showing cDNA sequence and the corresponding amino acid sequencefor AtNHX2.

FIG. 1(b) Shows the nucleic acid molecule that is SEQ ID NO:3 and thepolypeptide that is SEQ ID NO:4.

In a preferred embodiment, the figure shows isolated AtNHX4 cDNAencoding a Na⁺/H⁺ exchanger from Arabidopsis thaliana showing cDNAsequence and the corresponding predicted amino acid sequence for AtNHX4.

FIG. 1(c) Shows the nucleic acid molecule that is SEQ ID NO:5 and thepolypeptide that is SEQ ID NO:6. In a preferred embodiment, the figureshows isolated AtNHX5 cDNA encoding a Na⁺/H⁺ exchanger from Arabidopsisthaliana showing cDNA sequence and the corresponding predicted aminoacid sequence for AtNHX5.

FIG. 2 shows Western-blotting used to detect the expression in yeast ofboth the full and short length of AtNHX5.

FIG. 2A Shows 60 μg of tonoplast samples from Δnhx1+AtNHX5 yeast line(lane1) and Δnhx1+pYpGE15 vector line (lane2) blocked with anti X5-GSTfusion protein antibody displaying a 57 KDa band for the full ORFtransformants.

FIG. 2B Shows 60 μg of tonoplast samples from Δnhx1+AtNHX5-S yeast line(lane1) and Δnhx1+pYpGE15 vector line (lane2) blocked with anti X5-GSTfusion protein antibody, displaying a 40 KDa band for the short ORFtransformants.

FIG. 3 shows the salt tolerance test of different yeast transformants.On both A and B plates, equal amount of yeasts cells of differenttransformants were loaded at 10×, 100× and 1000× dilutions.

FIG. 3A shows different yeast transformants on 0 mM NaCl. From top tobottom: WT yeast transformed with empty pYpGE15 vector (WT+pYpGE15);Δnhx1 transformed with empty pYpGE15 vector (Δhx1+pYpGE15); Δnhx1transformed with AtNHX5-pYpGE15 construct (Δnhx1+AtNHX5-pYpGE15); andΔnhx1 transformed with the short version of AtNHX3 cloned in pYpGE15vector (Δnhx1+AtNHX5-S-pYpGE15).

FIG. 3B shows different yeast transformants on 200 mM NaCl. From top tobottom: WT yeast transformed with empty pYpGE15 vector (WT+pYpGE15);Δnhx1 transformed with empty pYpGE15 vector (Δhx1+pYpGE15); Δnhx1transformed with AtNHX5-pYpGE15 construct (Δnhx1+AtNHX5-pYpGE15); andΔnhx1 transformed with the short version of AtNHX3 cloned in pYpGE15vector (Δnhx1+AtNHX5-S-pYpGE15).

DETAILED DESCRIPTION OF THE INVENTION

Salt Tolerance Nucleic Acid Molecules and Polypeptides

The invention relates to nucleic acid molecules and polypeptides whichincrease salt tolerance in cells and plants. PNHX polypeptides are plantNa⁺/H⁺ transporter polypeptides that are capable of increasing andenhancing salt tolerance in a cell, preferably a plant cell. Thesetransporters (also referred to as exchangers, antiports or antiporters)extrude monovalent cations (preferably potassium ions or lithium ions,most preferably sodium ions) out of the cytosol. The cations arepreferably extruded into the vacuoles or extracellular space. Theaffinity for particular ions varies between transporters. The listedpreferences refer to the cations that are most likely to be abundant inthe cytosol and therefore most likely to be extruded. It is notnecessarily a reflection of transporter affinity for particular cations.The PNHX nucleic acid molecules which encode PNHX polypeptides areparticularly useful in producing transgenic plants which have increasedsalt tolerance compared to a wild type plant.

It will also be apparent that there are polypeptide and nucleic acidmolecules from other organisms, such as yeast, microorganisms, fish,birds or mammals, that are similar to PNHX polypeptides and nucleic acidmolecules. The entire group of Na⁺/H⁺ transporter polypeptides andnucleic acid molecules that are capable of increasing salt tolerance ina cell (including PNHX and AtNHX polypeptides and nucleic acidmolecules) are collectively referred to as (“TNHX polypeptides” and“TNHX nucleic acid molecules”). TNHX polypeptides are Na⁺/H⁺transporters that are capable of increasing salt tolerance in a cell,preferably a plant cell, because they extrude monovalent cations(preferably potassium ions or lithium ions, most preferably sodium ions)out of the cytosol.

The role of TNHX and PNHX nucleic acid molecules and polypeptides inmaintaining salt tolerance was not shown before this invention. Theability of these compounds to increase salt tolerance of transgenic hostcells (particularly plant cells) and transgenic plants compared to wildtype cells and plants was unknown.

PNHX and TNHX polypeptides need not necessarily have the primaryfunction of providing salt tolerance. All nucleotides and polypeptideswhich are suitable for use in the methods of the invention, such as thepreparation of transgenic host cells or transgenic plants, are includedwithin the scope of the invention. Genomic clones or cDNA clones arepreferred for preparation of transgenic cells and plants.

In a preferred embodiment, the invention relates to cDNAs encodingNa⁺/H⁺ exchangers from Arabidopsis thaliana. The cDNA sequences and thecorresponding amino acid sequences for AtNHX2, AtNHX4, and AtNHX5 arepresented in FIG. 1.

Function of Salt Tolerance Nucleic Acid Molecules

The polypeptides of the invention allow the extrusion of monovalentcations (preferably potassium ions or lithium ions, most preferablysodium ions) from the cytosol, which in this application preferablyrefers to the transport and accumulation of sodium ions into thevacuoles or into the extracellular space (outside of the cell), thusproviding the most important trait for salt tolerance in plants.Antiport polypeptides from organisms other than plants have showndifferent specificity for monovalent ions (e.g. D. G. Warnock, A. S.Pollock, “Sodium Proton Exchange in Epithelial Cells”, pages 77-90, inS. Grinstein ed. Sodium Proton Exchange, (1987, CRC Press, USA).) TNHXand PNHX transporters will also show different specificity betweentransporters. The nucleic acid molecules of the invention allow theengineering of salt tolerant plants by transformation of crops with thisnucleic acid molecule under the control of constitutively activepromoters or under the control of conditionally-inducible promoters. Theresulting expression or overexpression of these nucleic acid moleculesconfers increased salt tolerance in plants grown in soil, solid,semi-solid medium or hydroponically.

The PNHX Nucleic Acid Molecule and Polypeptide is Conserved in Plants

Sequence Identity

This is the first isolation of a nucleic acid molecule encoding a Na⁺/H⁺exchanger from plant species. It is widely known amongst those skilledin the art that Arabidopsis thaliana is a model plant for many plantspecies. Nucleic acid sequences having sequence identity to the AtNHXsequences are found in other plants, in particular halophytes such asBeta Vulgaris and Atriplex. Sequences from Arabidopsis thaliana andother plants are collectively referred to as “PNHX” nucleic acidsequences and polypeptides. We isolate PNHX nucleic acid molecules fromplants having nucleic acid molecules that are similar to those inArabidopsis thaliana, such as beet, tomato, rice, cucumber, radish andother plants as in Table 5 and using techniques described in thisapplication. The invention includes methods of isolating these nucleicacid molecules and polypeptides as well as methods of using thesenucleic acid molecules and polypeptides according to the methodsdescribed in this application, for example those used with respect toAtNHX.

Table 1 below shows several sequences with sequence identity andsequence similarity to the AtNHX polypeptides. Where polypeptides areshown, a suitable corresponding DNA encoding the polypeptide will beapparent. These sequences code for polypeptides similar to portions ofAtNHX polypeptides. The sequences in Table 2 are useful to make probesto identity full length sequences or fragments (from the listed speciesor other species). One skilled in the art would be able to design aprobe based on a polypeptide or peptide fragment. The invention includesnucleic acid molecules of about: 10 to 50 nucleotides, 50 to 200nucleotides, 200 to 500 nucleotides, 500 to 1000 nucleotides, 1000 to1500 nucleotides, 1500 to 1700 nucleotides, 1700 to 2000 nucleotides,2000 to 2500 nucleotides or at least 2500 nucleotides and which includeall or part of the sequences (or corresponding nucleic acid molecule) inTable 2. The invention also includes peptides and polypeptides of about:10 to 50 amino acids, 50 to 200 amino acids, 200 to 500 amino acids, 500to 750 amino acids or at least 750 amino acids which encode all or partof the polypeptides in Table 2 (wherein the polypeptide is producedaccording to a reading frame aligned with an AtNHX polypeptide).Possible modifications to these sequences will also be apparent. Thepolypeptide and nucleic acid molecules are also useful in researchexperiments or in bioinformatics to locate other sequences. The nucleicacid molecules and polypeptides preferably provide Na⁺/H⁺ transporteractivity and are capable of moving monovalent cations from the cytosolof the cell into vacuoles or the extracellular space (in thisapplication, extracellular space refers to the space outside a cell inan organism or the space outside a cultured cell).

TABLE 1 Organism GenBank Accession No. Yeast (S. pombe) 3850064 Yeast(Saccharomyces cervisae) 927695 Rice EST C 91832 Rice EST C 91861 RiceEST AV032544 Medicago Trunculata EST AA660573 Hordeum Vulgare STS L44032

As shown in Table 2 below, many nucleic acid molecules identified inArabidopsis thaliana have striking DNA sequence similarity to nucleicacid molecules encoding the homologous polypeptide in other plantspecies. Using the techniques described in this application and othersknown in the art, it will be apparent that the nucleic acid moleculeencoding the homologous Na⁺/H⁺ exchanger in other plant speciesincluding, but not limited to plants of agricultural and commercialinterest, will have DNA sequence identity (homology) at leastabout >17%, >20%, >25%, >35% to a DNA sequence shown in FIG. 1 (or apartial sequence thereof). Some plants species may have DNA with asequence identity (homology) at least about: >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to a DNAsequence in FIG. 1 (or a partial sequence thereof). The invention alsoincludes modified nucleic acid molecules from plants other thanArabidopsis thaliana which have sequence identity at leastabout: >17%, >20%, >25%, >35%, >50%, >60%, >70%, >80% or >90% morepreferably at least about >95%, >99% or >99.5%, to an AtNHX sequence inFIG. 1 (or a partial sequence thereof). Modified nucleic acid moleculesare discussed below. Preferably about 1, 2, 3, 4, 5, 6 to 10, 10 to 25,26 to 50 or 51 to 100, or 101 to 250 nucleotides or amino acids aremodified. Sequence identity is most preferably calculated as the numberof identical amino acid residues expressed as a percentage of the lengthof the shorter of the two sequences in a pairwise alignment. Thepairwise alignment is constructed preferably using the Clustal W programpreferably using the following parameter settings: fixed gap penalty=10,floating gap penalty=10, protein weight matrix=BLOSUM62.

The invention also includes nucleic acid molecules encoding polypeptideshaving sequence similarity taking into account conservative amino acidsubstitutions. Sequence similarity (and preferred percentages) arediscussed below.

It will be apparent that nucleic acid molecule encoding the homologousNa⁺/H⁺ exchanger in other species (preferably plants) including, but notlimited to plants of agricultural and commercial interest, willhybridize to all or part of a sequence in FIG. 1 (or a partial sequencethereof) under low, moderate (also called intermediate conditions) orhigh stringency conditions. Preferred hybridization conditions aredescribed below.

The invention includes the nucleic acid molecules from other plants aswell as methods of obtaining the nucleic acid molecules by, for example,screening a cDNA library or other DNA collection with a probe of theinvention (such as a probe comprising at least about: 10 or preferablyat least 15 or 30 nucleotides of AtNHX2, AtNHX4 or AtNHX5 and detectingthe presence of a TNHX or PNHX nucleic acid molecule. Another methodinvolves comparing the AtNHX sequences (eg in FIG. 1 to other sequences,for example using bioinfornatics techniques such as database searches oralignment strategies, and detecting the presence of a TNHX or PNHXnucleic acid molecule or polypeptide. The invention includes the nucleicacid molecule and/or polypeptide obtained according to the methods ofthe invention. The invention also includes methods of using the nucleicacid molecules, for example to make probes, in research experiments orto transform host cells or make transgenic plants. These methods are asdescribed below.

The polypeptides encoded by the homologous TNHX or PNHX nucleic acidmolecules in other species will have amino acid sequence identity. Thepreferred percentage of sequence identity for sequences of the inventionincludes sequences having identity of at least about: 30% to AtNHX1, 31%to AtNHX2, 36% to AtNHX3, and 36% to AtNHX4. Sequence identity may be atleast about: >20%, >25%, >28%, >30%, >35%, >40%, >50% to an amino acidsequence shown in FIG. 1 (or a partial sequence thereof). Somepolypeptides may have a sequence identity of at leastabout: >60%, >70%, >80% or >90%, more preferably at leastabout: >95%, >99% or >99.5% to an amino acid sequence in FIG. 1 (or apartial sequence thereof). Identity is calculated according to methodsknown in the art. Sequence identity is most preferably assessed by theClustal W program. The invention also includes modified polypeptidesfrom plants which have sequence identity at leastabout: >20%, >25%, >28%, >30%, >35%, >40%, >50%, >60%, >70%, >80%or >90% more preferably at least about >95%, >99% or >99.5%, to an AtNHXsequence in FIG. 1 (or a partial sequence thereof). Modifiedpolypeptides molecules are discussed below. Preferably about: 1, 2, 3,4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250nucleotides or amino acids are modified.

TABLE 2 Polypeptide DNA Plant Vacuolar H⁺-PPiase (vacuolarpyrophosphatase) Arabidopsis (Accession # 282878) 100%   100%   Beet(Accession # 485742) 88.7% 72.8% Tobacco (Accession # 1076627) 89.9%68.4% Rice (Accession # 1747296) 85%   70.4% Tonoplast IntrinsicPolypeptide (water channel) Arabidopsis (Accession # X63551) 100%  100%   Curcubita (Cucumber) (Accession # D45078) 66.5% 39.1% Raphanus(radish) (Accession # D84669) 56.7% 37.4% Helianthus (Accession #X95951) 50.4% 35.2% High Affinity Ammonium Transporter Arabidopsis(Accession # X75879) 100%   100%   Tomato (Accession # X95098) 73.5%62.9% Rice (Accession # AF001505) 66.6% 58.1%Nucleic Acid Molecules and Polypeptides Similar to AtNHX

Those skilled in the art will recognize that the nucleic acid moleculesequences in FIGS. 1(a), (b) and (c) are not the only sequences whichmay be used to provide increased salt tolerance in plants. The geneticcode is degenerate so other nucleic acid molecules which encode apolypeptide identical to an amino acid sequence in FIG. 1(a), (b) or (c)may also be used. The sequence of the other nucleic acid molecules ofthis invention may also be varied without changing the polypeptideencoded by the sequence. Consequently, the nucleic acid moleculeconstructs described below and in the accompanying examples for thepreferred nucleic acid molecules, vectors, and transformants of theinvention are merely illustrative and are not intended to limit thescope of the invention.

The sequences of the invention can be prepared according to numeroustechniques. The invention is not limited to any particular preparationmeans. For example, the nucleic acid molecules of the invention can beproduced by cDNA cloning, genomic cloning, DNA synthesis, polymerasechain reaction (PCR) technology, or a combination of these approaches([31] or Current Protocols in Molecular Biology (F. M. Ausbel et al.,1989).). Sequences may be synthesized using well known methods andequipment, such as automated synthesizers. Nucleic acid molecules may beamplified by the polymerase chain reaction. Polypeptides may, forexample, be synthesized or produced recombinantly.

Sequence Identity

The invention includes modified nucleic acid molecules with a sequenceidentity at least about: >17%, >20%, >30%, >40%, >50%, >60%, >70%, >80%or >90% more referably at least about >95%, >99% or >99.5%, to a DNAsequence in FIG. 1 (or a partial sequence thereof). Preferably about 1,2, 3, 4, 5, 6 to 10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250nucleotides or amino acids are modified. Identity is calculatedaccording to methods known in the art. Sequence identity is mostpreferably assessed by the Clustal W program. For example, if anucleotide sequence (called “Sequence A”) has 90% identity to a portionof the nucleotide sequence in FIG. 1(a), then Sequence A will beidentical to the referenced portion of the nucleotide sequence in FIG.1, except that Sequence A may include up to 10 point mutations, such asdeletions or substitutions with other nucleotides, per each 100nucleotide of the referenced portion of the nucleotide sequence in FIG.1. Nucleotide sequences functionally equivalent to the PNHX or AtNHXsequences can occur in a variety of forms as described below.Polypeptides having sequence identity may be similarly identified.

The polypeptides encoded by the homologous NHX, PHX Na⁺/H⁺ exchangenucleic acid molecule in other species will have amino acid sequenceidentity (also known as homology) at leastabout: >20%, >25%, >28%, >30%, >40% or >50% to an amino acid sequenceshown in FIG. 1 (or a partial sequence thereof). Some plants species mayhave polypeptides with a sequence identity (homology) of at leastabout: >60%, >70%, >80% or >90%, more preferably at leastabout: >95%, >99% or >99.5% to all or part of an amino acid sequence inFIG. 1 (or a partial sequence thereof). Identity is calculated accordingto methods known in the art. Sequence identity is most preferablyassessed by the Clustal W program. Preferably about: 1, 2, 3, 4, 5, 6 to10, 10 to 25, 26 to 50 or 51 to 100, or 101 to 250 nucleotides or aminoacids are modified.

The invention includes nucleic acid molecules with mutations that causean amino acid change in a portion of the polypeptide not involved inproviding salt tolerance and ion transport or an amino acid change in aportion of the polypeptide involved in providing salt tolerance so thatthe mutation increases or decreases the activity of the polypeptide.

Hybridization

Other functional equivalent forms of the AtNHX nucleic acid moleculesencoding nucleic acids can be isolated using conventional DNA-DNA orDNA-RNA hybridization techniques. These nucleic acid molecules and theAtNHX sequences can be modified without significantly affecting theiractivity.

The present invention also includes nucleic acid molecules thathybridize to one or more of the sequences in FIG. 1 (or a partialsequence thereof) or their complementary sequences, and that encodeexpression for peptides or polypeptides exhibiting substantiallyequivalent activity as that of an AtNHX polypeptide produced by the DNAin FIG. 1 or their variants. Such nucleic acid molecules preferablyhybridize to the sequences under low, moderate (intermediate), or highstringency conditions. (sec Sambrook et al. (Most recent edition)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.).

The present invention also includes nucleic acid molecules from anysource, whether modified or not, that hybridize to genomic DNA, cDNA, orsynthetic DNA molecules that encode the amino acid sequence of an AtNHXpolypeptide, or genetically degenerate forms, under salt and temperatureconditions equivalent to those described in this application, and thatcode for a peptide, polypeptide or polypeptide that has Na⁺/H⁺transporter activity. Preferably the polypeptide has the same or similaractivity as that of an AtNHX polypeptide. The nucleic acid molecules mayencode TNHX or PNHX polypeptides. A nucleic acid molecule describedabove is considered to be functionally equivalent to an AtNHX nucleicacid molecule (and thereby having Na⁺/H⁺ transporter activity) of thepresent invention if the polypeptide produced by the nucleic acidmolecule displays the following characteristics: the polypeptidemediates the proton-dependent sodium transport and sodium-dependentproton transport in intact cells, isolated organelles and purifiedmembrane vesicles. These sodium/proton movements should be higher(preferably at least about 50% higher and most preferably at least about100% higher) than the proton movements observed in the presence of abackground of potassium ions and/or other monovalent cations (i.e.rubidium, cesium, etc., but most preferably not lithium) (13,14).

The invention also includes nucleic acid molecules and polypeptideshaving sequence similarity taking into account conservative amino acidsubstitutions. Sequence similarity (and preferred percentages) arediscussed below.

Modifications to Nucleic Acid Molecule or Polypeptide Sequence

Changes in the nucleotide sequence which result in production of achemically equivalent or chemically similar amino acid sequences areincluded within the scope of the invention. Variants of the polypeptidesof the invention may occur naturally, for example, by mutation, or maybe made, for example, with polypeptide engineering techniques such assite directed mutagenesis, which are well known in the art forsubstitution of amino acids. For example, a hydrophobic residue, such asglycine can be substituted for another hydrophobic residue such asalanine. An alanine residue may be substituted with a more hydrophobicresidue such as leucine, valine or isoleucine. A negatively chargedamino acid such as aspartic acid may be substituted for glutamic acid. Apositively charged amino acid such as lysine may be substituted foranother positively charged amino acid such as arginine.

Therefore, the invention includes polypeptides having conservativechanges or substitutions in amino acid sequences. Conservativesubstitutions insert one or more amino acids which have similar chemicalproperties as the replaced amino acids. The invention includes sequenceswhere conservative substitutions are made that do not destroy Na⁺/H⁺transporter activity of the transporter polypeptide. Sequence similarityis preferably calculated as the number of similar amino acids in apairwise alignment expressed as a percentage of the shorter of the twosequences in the alignment. The pairwise alignment is preferablyconstructed using the Clustal W program, using the following parametersettings: fixed gap penalty=10, floating gap penalty=10, protein weightmatrix=BLOSUM62. Similar amino acids in a pairwise alignment are thosepairs of amino acids which have positive alignment scores defined in thepreferred protein weight matrix (BLOSUM62). The protein weight matrixBLOSUM62 is considered appropriate for the comparisons described here bythose skilled in the art of bioinformatics. (The reference for theclustal w program (algorithm) is Thompson, J. D., Higgins, D. G. andGibson, T. J. (1994) CLUSTAL W: improving the sensitivity of progressivemultiple sequence alignment through sequence weighting,positions-specific gap penalties and weight matrix choice. Nucleic AcidsResearch, 22:4673-4680; and the reference for BLOSUM62 scoring matrix isHenikoff, S. and Henikoff, J. G. (1993) Performance evaluation of aminoacid substitution matrices. Proteins, 7:49-61.)

Polypeptides comprising one or more d-amino acids are contemplatedwithin the invention. Also contemplated are polypeptides where one ormore amino acids are acetylated at the N-terminus. Those of skill in theart recognize that a variety of techniques are available forconstructing polypeptide mimetics with the same or similar desiredbiological activity (Na⁺/H⁺ transporter activity) as the correspondingpolypeptide compound of the invention but with more favorable activitythan the polypeptide with respect to solubility, stability, and/orsusceptibility to hydrolysis and proteolysis. See, for example, Morganand Gainor, Ann. Rep. Med. Chem., 24:243-252 (1989). Examples ofpolypeptide mimetics are described in U.S. Pat. No. 5,643,873. Otherpatents describing how to make and use mimetics include, for example in,U.S. Pat. Nos. 5,786,322, 5,767,075, 5,763,571, 5,753,226, 5,683,983,5,677,280, 5,672,584, 5,668,110, 5,654,276, 5,643,873. Mimetics of thepolypeptides of the invention may also be made according to othertechniques known in the art. For example, by treating a polypeptide ofthe invention with an agent that chemically alters a side group byconverting a hydrogen group to another group such as a hydroxy or aminogroup. Mimetics preferably include sequences that are either entirelymade of amino acids or sequences that are hybrids including amino acidsand modified amino acids or other organic molecules.

The invention also includes hybrid nucleic acid molecules andpolypeptides, for example where a nucleotide sequence from one speciesof plant is combined with a nucleotide sequence from another sequence ofplant, mammal or yeast to produce a fusion polypeptide. The inventionincludes a fusion protein having at least two components, wherein afirst component of the fusion protein comprises a polypeptide of theinvention, preferably a full length AtNHX polypeptide. The secondcomponent of the fusion protein preferably comprises a tag, for exampleGST, an epitope tag or an enzyme. The fusion protein may comprise lacZ.

The invention also includes polypeptide fragments of the polypeptides ofthe invention which may be used to confer salt tolerance if thefragments retain Na⁺/H⁺ transporter activity. The invention alsoincludes polypeptides fragments of the polypeptides of the inventionwhich may be used as a research tool to characterize the polypeptide orits activity. Such polypeptides preferably consist of at least 5 aminoacids. In preferred embodiments, they may consist of 6 to 10, 11 to 15,16 to 25, 26 to 50, 51 to 75,76 to 100 or 101 to 250 amino acids of thepolypeptides of the invention (or longer amino acid sequences). Thefragments preferably have sodium/proton transporter activity. Fragmentsmay include sequences with one or more amino acids removed, for example,C-terminus amino acids in an AtNHX sequence.

The invention also includes a composition comprising all or part of anisolated TNHX or PNHX (preferably AtNHX) nucleic acid molecule of theinvention and a carrier, preferably in a composition for planttransformation. The invention also includes a composition comprising anisolated TNHX or PNHX polypeptide (preferably AtNHX) and a carrier,preferably for studying polypeptide activity.

Recombinant Nucleic Acid Molecules

The invention also includes recombinant nucleic acid moleculescomprising a nucleic acid molecule of the invention and a promotersequence, operatively linked so that the promoter enhances transcriptionof the nucleic acid molecule in a host cell (the nucleic acid moleculesof the invention may be used in an isolated native gene or a chimericgene (for example, where a nucleic acid molecule coding region isconnected to one or more heterologous sequences to form a gene). Thepromoter sequence is preferably a constitutive promoter sequence or aninducible promoter sequence, operatively linked so that the promoterenhances transcription of the DNA molecule in a host cell. The promotermay be of a type not naturally associated with the cell. Transcriptionis enhanced with promoters known in the art such as the “Super-promoter”[20] or the 35S promoter of cauliflower mosaic virus [21].

Inducible promoters are also used. These include:

-   -   a) drought- and ABA-inducible promoters which may include        ABA-responsive elements [22,23]    -   b) heat shock-inducible promoters which may contain HSEs (heat        shock elements) as well as CCAAT box sequences [24]    -   e) salt-inducible promoters which may include AT an d PR        elements [25]    -   d) Copper-inducible promoter that includes ACE1binding sites        [26]    -   e) steroid-inducible promoter that includes the glucocorticoid        response element along with an expression vector coding for a        mammalian steroid receptor [27].

In addition, tissue specific expression is achieved with the use oftissue-specific promoters such as, the Fd (Ferredoxin) promoter thatmediates high levels of expression in green leaves [28] and peroxidasepromoter for root-specific expression [29]. These promoters vary intheir transcription initiation rate and/or efficiency.

A recombinant nucleic acid molecule for conferring salt tolerance mayalso contain suitable transcriptional or translational regulatoryelements. Suitable regulatory elements may be derived from a variety ofsources, and they may be readily selected by one with ordinary skill inthe art. Examples of regulatory elements include: a transcriptionalpromoter and enhancer or RNA polymerase binding sequence, a ribosomalbinding sequence, including a translation initiation signal.Additionally, depending on the vector employed, other genetic elements,such as selectable markers, may be incorporated into the recombinantmolecule. Markers facilitate the selection of a transformed host cell.Such markers include genes associated with temperature sensitivity, drugresistance, or enzymes associated with phenotypic characteristics of thehost organisms.

Nucleic acid molecule expression levels are controlled with atranscription initiation region that regulates transcription of thenucleic acid molecule or nucleic acid molecule fragment of interest in aplant, bacterial or yeast cell. The transcription initiation region maybe part of the construct or the expression vector. The transcriptioninitiation domain or promoter includes an RNA polymerase binding siteand an mRNA initiation site. Other regulatory regions that may be usedinclude an enhancer domain and a termination region. The regulatoryelements described above may be from animal, plant, yeast, bacterial,fungal, viral or other sources, including synthetically producedelements and mutated elements.

Methods of modifying DNA and polypeptides, preparing recombinant nucleicacid molecules and vectors, transformation of cells, expression ofpolypeptides are known in the art. For guidance, one may consult thefollowing U.S. Pat. Nos. 5,840,537, 5,850,025, 5,858,719, 5,710,018,5,792,851, 5,851,788, 5,759,788, 5,840,530, 5,789,202, 5,871,983,5,821,096, 5,876,991, 5,422,108, 5,612,191, 5,804,693, 5,847,258,5,880,328, 5,767,369, 5,756,684, 5,750,652, 5,824,864, 5,763,211,5,767,375, or 5,750,848. Many of these patents also provide guidancewith respect to experimental assays, probes and antibodies,transformation of host cells and regeneration of plants, which aredescribed below. These patents, like all other patents, publications(such as articles and Genbank publications) in this application, areincorporated by reference in their entirety.

Host Cells Including a Salt Tolerance Nucleic Acid Molecule

In a preferred embodiment of the invention, a plant or yeast cell istransformed with a nucleic acid molecule of the invention or a fragmentof a nucleic acid molecule and inserted in a vector.

Another embodiment of the invention relates to a method of transforminga host cell with a nucleic acid molecule of the invention or a fragmentof a nucleic acid molecule, inserted in a vector. The invention alsoincludes a vector comprising a nucleic acid molecule of the invention.The TNHX, PNHX and AtNHX nucleic acid molecules can be cloned into avariety of vectors by means that are well known in the art. Therecombinant nucleic acid molecule may be inserted at a site in thevector created by restriction enzymes. A number of suitable vectors maybe used, including cosmids, plasmids, bacteriophage, baculoviruses andviruses. Suitable vectors are capable of reproducing themselves andtransforming a host cell. The invention also relates to a method ofexpressing polypeptides in the host cells. A nucleic acid molecule ofthe invention may be used to transform virtually any type of plant,including both monocots and dicots. The expression host may be any cellcapable of expressing TNHX, PNHX, such as a cell selected from the groupconsisting of a seed (where appropriate), plant cell, bacterium, yeast,fungus, protozoa, algae, animal and animal cell.

Levels of nucleic acid molecule expression may be controlled withnucleic acid molecules or nucleic acid molecule fragments that code foranti-sense RNA inserted in the vectors described above.

Agrobacterium tumefaciens-mediated transformation,particle-bombardment-mediated transformation, direct uptake,microinjection, coprecipitation and electroporation-mediated nucleicacid molecule transfer are useful to transfer a Na⁺/H⁺ transporternucleic acid molecule into seeds (where appropriate) or host cells,preferably plant cells, depending upon the plant species. The inventionalso includes a method for constructing a host cell capable ofexpressing a nucleic acid molecule of the invention, the methodcomprising introducing into said host cell a vector of the invention.The genome of the host cell may or may not also include a functionalTNHX or PNHX gene. The invention also includes a method for expressing aTNHX or PNHX transporter polypeptide in the host cell or a plant, plantpart, seed or plant cell of the invention, the method comprisingculturing the host cell under conditions suitable for gene expression.The method preferably also includes recovering the expressed polypeptidefrom the culture.

The invention includes the host cell comprising the recombinant nucleicacid molecule and vector as well as progeny of the cell. Preferred hostcells are fungal cells, yeast cells, bacterial cells, mammalian cells,bird cells, reptile cells, amphibious cells, microorganism cells andplant cells. Host cells may be cultured in conventional nutrient media.The media may be modified as appropriate for inducing promoters,amplifying genes or selecting transformants. The culture conditions,such as temperature, composition and pH will be apparent. Aftertransformation, transformants may be identified on the basis of aselectable phenotype. A selectable phenotype can be conferred by aselectable marker in the vector.

Transgenic Plants and Seeds

Plant cells are useful to produce tissue cultures, seeds or wholeplants. The invention includes a plant, plant part, seed, or progenythereof including a host cell transformed with a PNHX nucleic acidmolecule. The plant part is preferably a leaf, a stem, a flower, a root,a seed or a tuber.

The invention includes a transformed (transgenic) plant having increasedsalt tolerance, the transformed plant containing a nucleic acid moleculesequence encoding for Na⁺/H⁺ transporter polypeptide activity and thenucleic acid molecule sequence having been introduced into the plant bytransformation under conditions whereby the transformed plant expressesa Na⁺/H⁺ transporter in active form.

The methods and reagents for producing mature plants from cells areknown in the art. The invention includes a method of producing agenetically transformed plant which expresses PNHX or TNHX polypeptideby regenerating a genetically transformed plant from the plant cell,seed or plant part of the invention. The invention also includes thetransgenic plant produced according to the method. Alternatively, aplant may be transformed with a vector of the invention.

The invention also includes a method of preparing a plant with increasedsalt tolerance, the method comprising transforming the plant with anucleic acid molecule which encodes a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide encoding a Na⁺/H⁺transporter polypeptide capable of increasing salt tolerance in a cell,and recovering the transformed plant with increased salt tolerance. Theinvention also includes a method of preparing a plant with increasedsalt tolerance, the method comprising transforming a plant cell with anucleic acid molecule which encodes a TNHX transporter polypeptide, aPNHX transporter polypeptide or a polypeptide encoding a Na⁺/H⁺transporter polypeptide capable of increasing salt tolerance in a cell,and producing the transformed plant with increased salt tolerance.

Overexpression of Na⁺/H⁺ exchangers leads to an improved ability of thetransgenic plants to uptake more monovalent cations from the growthmedia (soil) leading to an increased or enhanced tissue expansion.Therefore, the invention also relates to methods of producing or growingplants with increased tissue expansion (this could be manifested asenhanced size, growth or growth potential and may appear as increased orenhanced root, crown, shoot, stem, leaf, flower size or abundance incomparison to a wild type plant). The methods of preparing plants thathave increased tissue expansion are the same as the methods forpreparing a plant with increased salt tolerance described in thisapplication (or the methods are easily adapted, to the extent that thereis a difference in the methods).

The plants whose cells maybe transformed with a nucleic acid molecule ofthis invention and used to produce transgenic plants include, but arenot limited to the following:

Target plants.

Group I (Transformable Preferably Via Agrobacterium tumefaciens)

-   Arabidopsis-   Potato-   Tomato-   Brassica-   Cotton-   Sunflower-   Strawberries-   Spinach-   Lettuce-   Rice    Group II (Transformable Preferably Via Biolistic Particle Delivery    Systems (Particle Bombardment)-   Soybean-   Rice-   Corn-   Wheat-   Rye-   Barley-   Atriplex-   Salicornia

The nucleic acid molecule may also be used with other plants such asoat, barley, hops, sorgum, alfalfa, sunflower, alfalfa, beet, pepper,tobacco, melon, squash, pea, cacao, hemp, coffee plants and grape vines.Trees may also be transformed with the nucleic acid molecule. Such treesinclude, but are not limited to maple, birch, pine, oak and poplar.Decorative flowering plants such as carnations and roses may also betransformed with the nucleic acid molecule of the invention. Plantsbearing nuts such as peanuts may also be transformed with the salttolerance nucleic acid molecule. A list of preferable plants is in Table5.

In a preferred embodiment of the invention, plant tissue cells orcultures which demonstrate salt tolerance are selected and plants whichare salt tolerant are regenerated from these cultures. Methods ofregeneration will be apparent to those skilled in the art (see Examplesbelow, also). These plants may be reproduced, for example by crosspollination with a plant that is salt tolerant or a plant that is notsalt tolerant. If the plants are self-pollinated, homozygous salttolerant progeny may be identified from the seeds of these plants, forexample by growing the seeds in a saline environment, using geneticmarkers or using an assay for salt tolerance. Seeds obtained from themature plants resulting from these crossings may be planted, grown tosexual maturity and cross-pollinated or self-pollinated.

The nucleic acid molecule is also incorporated in some plant species bybreeding methods such as back crossing to create plants homozygous forthe salt resistance nucleic acid molecule.

A plant line homozygous for the salt tolerance nucleic acid moleculemaybe used as either a male or female parent in a cross with a plantline lacking the salt tolerance nucleic acid molecule to produce ahybrid plant line which, is uniformly heterozygous for the nucleic acidmolecule. Crosses between plant lines homozygous for the salt resistancenucleic acid molecule are used to generate hybrid seed homozygous forthe resistance nucleic acid molecule.

The nucleic acid molecule of the invention may also be used as a markerin transformation experiments with plants. A salt sensitive plant may betransformed with a salt tolerance nucleic acid molecule and a nucleicacid molecule of interest which are linked. Plants transformed with thenucleic acid molecule of interest will display improved growth in asaline environment relative to the non-transformed plants.

Fragments/Probes

Preferable fragments (fragments are also referred to as polypeptidefragments or peptide fragments) include 10 to 50, 50 to 100, 100 to 250,250 to 500, 500 to 1000, 1000 to 1500, or 1500 or more nucleotides of anucleic acid molecule of the invention. A fragment may be generated byremoving a single nucleotide from a sequence in FIG. 1 or 5 (or apartial sequence thereof). Fragments may or may not have Na⁺/H⁺transporter activity.

The nucleic acid molecules of the invention (including a fragment of asequence in FIG. 1 (or a partial sequence thereof) (such as SEQ ID NO:1,SEQ ID NO:3, or SEQ ID NO:5) can be used as probes to detect nucleicacid molecules according to techniques known in the art (for example,see U.S. Pat. Nos. 5,792,851 and 5,851,788). The probes may be used todetect nucleic acid molecules that encode polypeptides similar to thepolypeptides of the invention. For example, a probe having at leastabout 10 bases will hybridize to similar sequences under stringenthybridization conditions (Sambrook et al. 1989, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor).

The invention includes oligonucleotide probes made from the AtNHXsequences described in this application or other nucleotide sequences ofthe invention. The probes may be about 10 to 30 or 15 to 30 nucleotidesin length and are preferably at least 30 or more nucleotides. Apreferred probe is 5′-TTCTTCATATATCTTTTGCCACCC-3′ (SEQ ID NO: 7) (codingfor the amiloride binding domain) or at least about 10 or 15 nucleotidesof this sequence. The invention also includes an oligonucleotideincluding at least 30 consecutive nucleotides of an AtNHX molecule inFIG. 1 or 5 (or a partial sequence thereof). The probes are useful toidentify nucleic acids encoding AtNHX polypeptides and proteins otherthan those described in the application, as well as peptides,polypeptides, and proteins have Na⁺/H⁺ transporter activity andpreferably functionally equivalent to AtNHX. The oligonucleotide probesare capable of hybridizing to one or more of the sequences shown in FIG.1 (or a partial sequence thereof) or the other sequences of theinvention under low, moderate or high stringency hybridizationconditions. A nucleotide sequence encoding a polypeptide of theinvention may be isolated from other organisms by screening a libraryunder low, moderate or high stringency hybridization conditions with adetectable probe (e.g. a labeled probe). The activity of the polypeptideencoded by the nucleotide sequence may be assessed by cloning andexpression of the DNA. After the expression product is isolated, thepolypeptide is assayed for Na⁺/H⁺ transporter activity as described inthis application.

Functionally equivalent AtNHX, TNHX or PNHX nucleic acid molecules fromother cells, or equivalent AtNHX, TNHX or PNHX -encoding cDNAs orsynthetic DNAs, can also be isolated by amplification using PolymeraseChain Reaction (PCR) methods. Oligonucleotide primers, includingdegenerate primers, based on the amino acid sequence of the sequences inFIG. 1 (or a partial sequence thereof) can be prepared and used inconjunction with PCR technology employing reverse transcriptase toamplify functionally equivalent DNAs from genomic or cDNA libraries ofother organisms. Alternatively, the oligonucleotides, includingdegenerate nucleotides, can be used as probes to screen cDNA libraries.

Thus, the invention includes an oligonucleotide probe comprising all orpart of a nucleic acid in FIG. 1 (or a partial sequence thereof), or acomplementary strand thereof. The probe is preferably labeled with adetectable marker. The invention also includes an oligonucleotidecomprising at least 10, 15 or 30 nucleotides capable of specificallyhybridizing with a sequence of nucleic acids of the nucleotide sequenceset forth in FIG. 1 (or a partial sequence thereof). The invention alsoincludes a single strand DNA primer for amplification of PNHX nucleicacid, wherein the primer is selected from a nucleic acid sequencederived from a nucleic acid sequence in FIG. 1 (or a partial sequencethereof).

The invention also includes a method for identifying nucleic acidmolecules encoding a TNHX, PNHX or AtNHX polypeptide. Techniques forperforming the methods are described in, for example, U.S. Pat. Nos.5,851,788 and 5,858,719. A preferred method includes contacting a samplecontaining nucleic acids with an oligonucleotide, wherein saidcontacting is effected under low, moderate or high stringencyhybridization conditions, and identifying nucleic acids which hybridizethereto. Hybridization forms a hybridization complex. The presence of acomplex correlates with the presence of a nucleic acid molecule encodingTNHX, plant PNHX polypeptide or AtNHX in the sample. In a preferredmethod, the nucleic acid molecules are amplified by the polymerase chainreaction prior to hybridization.

Kits

The invention also includes a kit for conferring increased salttolerance to a plant or a host cell including a nucleic acid molecule ofthe invention (preferably in a composition for the invention) andpreferably reagents for transforming the plant or host cell.

The invention also includes a kit for detecting the presence of a TNHXor a PNHX nucleic acid molecule, comprising at least one ofoligonucleotide of the invention. Kits may be prepared according toknown techniques, for example, see U.S. Pat. Nos. 5,851,788 and5,750,653.

Antibodies

The invention includes an isolated antibody immunoreactive with apolypeptide of the invention (see Example 1). The antibody may belabeled with a detectable marker or unlabeled. The antibody ispreferably a monoclonal antibody or a polyclonal antibody. TNHX, PN orAtNHX antibodies can be employed to screen organisms containing TNHX,PNHX or AtNHX polypeptides. The antibodies are also valuable forimmunopurification of polypeptides from crude extracts.

The isolated antibody is preferably specifically reactive with a TNHX orPNHX transporter, preferably an AtNHX transporter. The transporter ispreferably encoded by a nucleic acid molecule in FIG. 1 (or moleculesthat hybridize to a molecule in FIG. 1 under low, moderate or highstringency hybridization conditions or molecules having at least about:17%, at least 20%, at least 25%, or at least 35% sequence identity (orthe other preferred percentages of identity or sequence similaritydescribed above) to a molecule in FIG. 1 or 5 (or a partial sequencethereof). The transporter is preferably a polypeptide in FIG. 1 (orpolypeptides having at least about: 28%, 35% sequence identity (or theother preferred percentages of identity or sequence similarity describedabove) to a polypeptide in FIG. 1 or 5 (or a partial sequence thereof).The antibody preferably does not cross-react with other transporterpolypeptides. The antibody is preferably specifically reactive with apolypeptide having an amino acid sequence encoded by a nucleic acidmolecule set forth in FIG. 1 or 5 (or a partial sequence thereof).

Examples of the preparation and use of antibodies are provided in U.S.Pat. Nos. 5,792,851 and 5,759,788. For other examples of methods of thepreparation and uses of monoclonal antibodies, see U.S. Pat. Nos.5,688,681, 5,688,657, 5,683,693, 5,667,781, 5,665,356, 5,591,628,5,510,241, 5,503,987, 5,501,988, 5,500,345 and 5,496,705. Examples ofthe preparation and uses of polyclonal antibodies are disclosed in U.S.Pat. Nos. 5,512,282, 4,828,985, 5,225,331 and 5,124,147.

The invention also includes methods of using the antibodies. Forexample, the invention includes a method for detecting the presence ofTNHX, PNHX or AtNHX transporter polypeptide, by: a) contacting a samplecontaining one or more polypeptides with an antibody of the inventionunder conditions suitable for the binding of the antibody topolypeptides with which it is specifically reactive; b) separatingunbound polypeptides from the antibody; and c) detecting antibody whichremains bound to one or more of the polypeptides in the sample.

Research Tool

Cell cultures, seeds, plants and plant parts transformed with a nucleicacid molecule of the invention are useful as research tools. Forexample, one may obtain a plant cell (or a cell line, such as animmortalized cell culture or a primary cell culture) that does notexpress AtNHX1, insert an AtNHX1 nucleic acid molecule in the cell, andassess the level of AtNHX1 expression and activity. Alternatively, PNHXnucleic acid molecules may be overexpressed in a plant that expresses aPNHX nucleic acid molecule. In another example, experimental groups ofplants may be transformed with vectors containing different types ofPNHX nucleic acid molecules (or PNHX nucleic acid molecules similar toPNHX or fragments of PNHX nucleic acid molecules) to assess the levelsof protein produced, its functionality and the phenotype of the plants(for example, phenotype in saline soil). The polypeptides are alsouseful for in vitro analysis of TNHX, PNHX or AtNHX activity orstructure. For example, the polypeptides produced can be used formicroscopy or X-ray crystallography studies.

The TNHX, PNHX or AtNHX nucleic acid molecules and polypeptides are alsouseful in assays. Assays are useful for identification and developmentof compounds to inhibit and/or enhance polypeptide function directly.For example, they are useful in an assay for evaluating whether testcompounds are capable of acting as antagonists for PNHX polypeptides by:(a) culturing cells containing: a nucleic acid molecule which expressesPNHX polypeptides (or polypeptides having PNHX or Na⁺/H⁺ activity)wherein the culturing is carried out in the presence of: increasingconcentrations of at least one test compound whose ability to inhibittransport activity of PNHX polypeptide is sought to be determined, and afixed concentration of salt; and (b) monitoring in the cells the levelof salt transported out of the cytosol as a function of theconcentration of the test compound, thereby indicating the ability ofthe test compound to inhibit PNHX transporter activity. Alternatively,the concentration of the test compound may be fixed and theconcentration of salt may be increased.

Another experiment is an assay for evaluating whether test compounds arecapable of acting as agonists for PNHX polypeptide characterized bybeing able to transport salt across a membrane, (or polypeptides havingPNHX or Na^(+/H) ⁺ transporter activity) by (a) culturing cellscontaining: a nucleic acid molecule which expresses PNHX polypeptide or(or polypeptides having PNHX activity) thereof, wherein said culturingis carried out in the presence of: fixed concentrations of at least onetest compound whose ability to increase or enhance salt transportactivity of PNHX polypeptide is sought to be determined, and anincreasing concentration of salt; and (b) monitoring in the cells thelevel of salt transported out of the cytosol as a function of theconcentration of the test compound, thereby indicating the ability ofthe test compound compound to increase or enhance PNHX polypeptideactivity. Alternatively, the concentration of the test compound may befixed and the concentration of salt may be increased. Suitable assaysmay be adapted from, for example, U.S. Pat. No. 5,851,788. It isapparent that TNHX and AtNHX may also be used in assays.

Bioremediation

Soils containing excessive salt may be unable to grow plants in a mannersuitable for agriculture. The invention includes a method for removingsalt from a growth medium, comprising growing a plant transformed with anucleic acid molecule of the invention and expressing a salt toleranceNa⁺/H⁺ transporter polypeptide in the growth medium for a time periodsufficient for the plant root to uptake and accumulate salt in the rootor shoot biomass. The growth medium may be a solid medium, semi-solidmedium, liquid medium or a combination thereof. It may include soil,sand, sludge, compost, or artificial soil mix. The shoot (leaf or stem)or and root biomass may be harvested. Preferably, a sufficient portionof the shoot biomass is not harvested and is left in the growth media topermit continued plant growth.

Using Exogenous Agents in Combination with a Vector

The nucleic acid molecules of the invention may be used with othernucleic acid molecules that relate to salt tolerance, for example,osmoregulant genes. Host cells or plants may be transformed with thesenucleic acid molecules. Osmoregulants are disclosed, for example, inU.S. Pat. Nos. 5,563,324 and 5,639,950. The following Examples areintended to illustrate and assist in the further understanding of theinvention. Particular materials employed, species, conditions and thelike are not intended to limit the reasonable scope of the invention.

EXAMPLE 1

Preparation of Polyclonal and Monoclonal Antibodies.

Hydropathy profiles of the Arabidopsis Na⁺/H⁺ antiport revealed arelatively hydrophilic domain (at the C-terminus) with possibleregulatory functions. The C-terminus was sub-cloned into the pGEX—2TKvector (Pharmacia) to allow the overexpression of the C-terminuspolypeptide as a GST-fusion polypeptide in E. coli. The fusionpolypeptide was purified by glutathione-affinity chromatography and usedas an antigen in rabbits to obtain polyclonal antibodies [30].

Monoclonal antibodies are prepared in mice hybridomas according toestablished techniques [30] using the C-terminus polypeptide asdescribed above. Polyclonal and monoclonal antibodies raised againstother regulatory regions of the Arabidopsis Na⁺/H⁺ antiport are alsoobtained as described above. The invention includes the antibodies andthe hybridoma which secretes the monoclonal antibodies.

EXAMPLE 2

Identification of Homologous Nucleic Acid Molecules From Other PlantSpecies, Preferably Salt Tolerant Species.

Several experimental approaches are used to identify homologous nucleicacid molecules from salt tolerant species. a) We screen cDNA and genomiclibraries from sugar beets (a moderate salt-tolerant crop, also known asred beet) under low-stringency conditions with an Arabidopsis Na⁺/H⁺antiport cDNA as a probe [31]; b) We apply PCR techniques usingdegenerate oligonucleotide primers designed according to the conservedregions of the Arabidopsis Na⁺/H⁺ antiport c) We screen cDNA expressionlibraries from different plants (salt-tolerant and salt-sensitive) usingantibodies raised against an Arabidopsis Na⁺/H⁺ antiport [31]. We alsouse bioinformnatics techniques to identify nucleic acid molecules. Theinvention includes methods of using such a nucleic acid molecule, forexample to express a recombinant polypeptide in a transformed cell.

The techniques described above for isolating nucleic acid molecules fromArabidopsis and sugar beet are used to isolate a salt tolerance nucleicacid molecule from Atriplex and other plants.

EXAMPLE 3

Overexpression of the PNHX Transporter, Preferably ArabidopsisTransporter (AtNHX).

The Na⁺/H⁺ antiport is expressed in Arabidopsis plants, although thewild type plants show impaired growth at NaCl concentrations higher than75 mM. The Na⁺/H⁺ antiport is overexpressed in these plants in order toimprove their tolerance to high salt concentrations. A full length cDNA(preferably coding for the AtNHX1 polypeptide (AtNHX2, AtNHX3 or AtNHX4)cloned from an Arabidopsis thaliana (Columbia) seedling cDNA library isligated into a pB1NS1 vector [33]. This vector contains a constitutivelystrong promotor (“super-promotor” [20]). Also, T-DNA vectors (pBECKS)are used [34]. Constructs containing the AtNHX1 cDNA with the fullNa⁺/H⁺ antiport open reading frame in a sense orientation were selectedby colony hybridization using the AtNHX1 as a probe and byrestriction-digest analysis using Bg1II restriction endonuclease. Theseconstructs are used to transform Agrobacterium tumefaciens, and thesetransformed Agrobacterium tumefaciens are used for transformation ofArabidopsis plants. The Agrobacterium for inoculation is grown at 28° C.in a medium containing 5 g/l Bacto Beef Extract, 5 g/l Bacto-Peptone, 1g/l Bacto Yeast Extract, 240 mg MgSO₄ and 5 g/l sucrose. The pH will beadjusted to 7.2 with NaOH.

Arabidopsis seeds are washed and surface-sterilized in 5% (w/v) sodiumhypochlorite containing 0.15% (v/v) Tween-20. The seeds are rinsedthoroughly with sterile distilled water. Seed aliquots are dispensed inflasks containing 45 ml of cocultivation medium (MS salts, 100 mMsucrose, 10 mg/l thiamine, 0.5 mg/l pyridoxine, 0.5 mg/l nicotinic acid,100 mg/l inositol and the pH adjusted to 6.0 with KOH. The flasks areincubated at 22° C. under constant rotation (190 rpm) and constantlight. After 10-18 h (time needed to break clumps of seeds) 5 ml of logphase of Agrobacterium (OD₆₀₀=0.75) carrying the AtNHX1 construct areadded. Twenty-four hours following the inoculation, the seeds are driedby filtration and sown into pre-soaked vermiculite. The flats containingthe seeds are irrigated as required with a half-Hoagland solution. Theflats are covered with plastic to prevent desiccation and maintained atlow artificial illumination. After 3 days the flats are transferred tothe greenhouse (the plastic cover removed) under a 16/8 day/night cycle.Supplementary light is provided by high pressure sodium vapor lights.Seven weeks after sowing, the plants are dried thoroughly and the seeds(T2) harvested. Transformation efficiency is estimated by plating100,000 seeds (approximately 2.5 g of seeds) on agar plates containing50 mg/l kanamycin in a medium containing 1% (w/v) sucrose, 0.8 (w/v)agar, MS salts and a pH 6.0 adjusted with KOH. The plates aretransferred to a growth room at 25° C. under continuous light. After 10days the kanamycin-resistant seedlings are transferred to new growthmedium for 2 weeks and then transferred to small pots containingvermiculite. At senescence (8 weeks) the seeds are collected from singleplants (T3). These seeds are germinated and used to assess salttolerance of the transgenic plants.

EXAMPLE 4

Overexpression of TNHX or PNHX in Other Plants.

In a preferred method, overexpression of PNHX, preferably AtNHX2,AtNHX4, or AtNHX5, in a number of plants (potato, tomato, brassica,cotton, sunflower, strawberries, spinach, lettuce, rice, soybean, corn,wheat, rye, barley, atriplex, salicornia, and others) is achieved byAgrobacterium tumefaciens-based transformation and/or particlebombardment (AtNHX2, AtNHX4, AtNHX5 are also useful in this example).The full length cDNA (coding for the AtNHX2, 4 or 5) is ligated into thePBINS1 vector or pBECKS (as described above) and these constructs areused to transform Agrobacterium tumefaciens strain LBA4404.Agrobacterium used for inoculation is grown as described above. Culturedcells (callus), leaf explants, shoot and root cultures are used astargets for transformation. The targeted tissues are co-cultivated withthe bacteria for 1-2 days. Afterwards, the tissue is transferred to agrowth media containing kanamycin. After one week the tissue istransferred to a regeneration medium containing MS salts, 1% sucrose,2.5 mg/l 3-benzyladenine, 1 mg/l zeatin, 0.75% agar and kanamycin.Weekly transfers to fresh regeneration media are performed.

In another preferred embodiment, overexpression constructs carrying theAtNHX2, 4 or 5 cDNAs are introduced into an electro-competentAgrobacterium tumefaciens (LBA4404) by electroporation. The Agrobacteriaare plated on LB plates containing 50 mg/L kanamycin and grown for 2days at 30° C. to select for bacteria carrying the overexpressionconstructs. One liter liquid LB+kanamycin (50 mg/L) is inoculated with asingle Agrobacterium colony selected from the LB (kanamycin 50 mg/L)plates. The culture is grown to a minimum of OD=1 (600 nm) for 2-3 days.The Agrobacteria are then pelleted and resuspended in 1 L infiltrationmedium (IM-0.5×MS salts; 0.5 g/L MES; 5% sucrose; 0.03% Silwet L-77).Flowering Arabidopsis plants with primary bolts reaching ˜15 cm are usedfor the transformation procedure (T1). Pots of Arabidopsis plants aredunked into the IM solution containing the Agrobacteria and leftsubmerged for 2-6 minutes. The same procedure can be repeated after 8-12days on the same plants. Plants are allowed to senesce, the plants aredried thoroughly and the seeds harvested. Seeds are plated on agarplates containing 25 mg/L kanamycin in a medium containing MS salts,0.8% (w/v) agar adjusted to pH 6.0 with KOH. The plates are transferredto a growth room at 25° C. under continuous light. After 10 days thekanamycin-resistant seedlings (T2) are transferred to small potscontaining vermiculite. At senescence (˜8 weeks) the seeds are collectedfrom single plants and plated on agar plates containing MS salts and 25mg/L kanamycin. After 10 days the kanamycin-resistant seedlings (T3) aretransferred to small pots containing vermiculite. Seeds produced bythese plants are germinated and used to assess salt tolerance of thetransgenic plants. A biolistic particle delivery system (particlebombardment) is also used for the overexpression of NHX (AtNHX2, AtNHX4,or AtNHX5 are useful for this example). Constructs made using a plasmidvector preferably carrying a constitutive promoter, the AtNHX2, 4 or 5open reading frame in a sense orientation and a NOS termination site areused. Plasmid DNA is precipitated into 1.25 mg of 1-2 μm gold particlesusing 25 μl of 2.5 M CaCl₂ and 10 μl of 0.1 M thiamine (free base).DNA-coated particles are washed with 125 1 μl of 100% ethanol and thenresuspended in 30 μl ethanol. The samples are sonicated to obtain anefficient dispersion, and the samples are aliquoted to obtain deliverydisks containing 3 μg of DNA each. Particle bombardment is optimizedaccording to the specific tissue to be transformed. Tissue samples areplaced in Petri dishes containing 4.5 g/l basal MS salts, 1 mg/lthiamine, 10 mg/l myoinositol, 30 g/l sucrose, 2.5 mg/l amphotericin and10 mM K₂HPO₄ at pH 5.7. After bombardment the petri dishes are incubatedfor 18-24 hours. Tissue is regenerated in plates with growth mediacontaining the selective marker. Rooting is initiated and transformedplants are grown under optimal growth conditions in growth chambers.After 2-4 weeks the seedlings are transferred to new growth medium for 2weeks and then transferred to small pots containing vermiculite. Atsenescence the seeds are collected from single plants. These seeds aregerminated and used to assess salt tolerance of the transgenic plants.

EXAMPLE 5

Overexpression of AtNHX2, 4 or 5 Homologs in Other Plants.

Overexpression of AtNHX2, 4 or 5 homologs from other plant species,preferably salt tolerant species (i.e., sugar beet) in other plants(potato, tomato, brassica, cotton, sunflower, strawberries, spinach,lettuce, rice, soybean, corn, wheat, rye, barley, atriplex, salicornia,and others) is achieved by Agrobacterium tumefaciens-basedtransformation and/or particle bombardment as described above (inExamples 3 and 4). Regeneration of the transformed plants is performedas described in Examples 3 and 4.

EXAMPLE 6

Expression of AtNHX2, 4 or 5, Homologs and Derivatives in Saccharomycescerevisiae.

Expression of TNHX or PNHX, preferably AtNHX1, AtNHX2, 4 or 5 homologsand derivatives in yeast is useful to assess and characterize thebiochemical properties of the recombinant and native polypeptides.Expression in yeast also facilitates the study of interactions betweenAtNHX1, its homologs and derivatives with regulatory polypeptides. Wehave made conditional expression constructs by ligating the codingregion of the AtNHX2, 4 or 5 cDNA into two vectors, pYES2 (Invitrogen)and pYEP434 [35]. Both constructs provide galactose-inducibleexpression, but pYES2 has a URA3 selectable marker while pYEP434 hasLEU2 as a selectable marker. Transformation by lithium acetate [36],1994), is followed by selection on solid media containing amino acidsappropriate for the selection of cells containing the transformationvector. For integrative transformation, the YXplac series of vectors forintegrative transformation are used [37].

EXAMPLE 7

Molecular Characterization and Functional Analysis of Na⁺/H⁺ Exchangersfrom Arabidopsis and Other Plants, Preferably Salt-tolerant (halophytes)Plants.

We do molecular and biochemical characterization of the different Na⁺/H⁺exchangers from Arabidopsis and other plants, preferably salt tolerantplants (halophytes). We determine the expression patterns of thedifferent Arabidopsis putative exchangers. Using Northern blot analysiswith isoform-specific cDNA probes under high stringency conditions andstandard molecular biology protocols, we determine thetissue-specificity, developmental and salt-inducibility gene expressionprofiles of each isoform.

We employ common molecular biology procedures to isolate Na⁺/H⁺exchangers from other plants as described below, in particularhalophytes (such as Beta vulgaris, Atriplex, Messembryanthemumchrystalinum, etc.). We designed degenerate oligonucleotide PCR primers,based upon highly conserved regions within Na⁺/H⁺ exchangers (one withinthe amiloride-binding domain, and another within a region about 200amino acid residues further downstream) from Arabidopsis, yeast,mammals, and C. elegans, to generate a 600-1,000 by DNA fragments byPCR. Sequencing of these products revealed significant homology toAtNHX1 and they are therefore being used as a probe to screen thedifferent halophyte cDNA libraries to isolate the full-length cDNAs bystandard methods. We use the nucleic acid molecules obtained in thisprocedure in methods of producing transgenic host cells and plants asdescribed above.

We have subcloned unique regions from AtNHX1, AtNHX2 and AtNHX3 isoformsinto a prokaryotic expression vector (pGEX2TK, Pharmacia) for theproduction of recombinant GST-fusion proteins that are being used forthe generation of isoform-specific polyclonal antibodies in rabbits.Briefly, sequence-specific oligonucleotides, with 5′ BamHI (sensestrand) and 3′ EcoRI (antisense strand) flanking restriction sites, wereused for PCR-mediated amplification of the unique (partial) codingregions from each isoform, and the digested PCR products were ligatedinto EcoRI/BamHI-digested pGEX2TK vector. pGEX2TK plasmids containingthe inserts corresponding to each AtNHX isoform were sequenced on bothstrands to verify the fidelity of the PCR reaction and were used forexpression and purification of the recombinant GST-fusion proteins in E.Coli (BL21pLysS) as per manufacturers instructions (Pharmacia). Wefollow an identical procedure to that described above to producerecombinant halophyte-PNHX GST-fusion protein in E. coli. Antibodiesagainst the fusion proteins are produced in rabbits by standardprocedures and their isoform-specificity are confirmed by westernblotting using the different GST-fusion proteins. The antibodies areused in conjunction with subcellular membrane fractions (prepared fromsucrose density gradients) [15] from various Arabidopsis and other planttissues, preferably halophyte tissues and western blots to determine thesubcellular localization of each Na⁺/H⁺ exchanger isoform. Theselocalization studies assign functions to the various isoforms.

EXAMPLE 8

Biochemical Characterization and Functional Analysis of Na⁺/H⁺exchangers from Arabidopsis and Other Plants, Preferably salt-tolerant(Halophytes) plants.

Biochemical characterization of the Na⁺/H⁺ exchanger isoforms isperformed in (i) heterologous eukaryotic expression systems (baculovirusexpression system in Sf9 insect cells, transgenic yeast); and in (ii)transgenic plants.

The use of heterologous expression systems allows the fastcharacterization of the kinetic properties of each exchanger isoform(K_(m), V_(max), ion specificity). Baculo-virusinfected Sf9 cells haveproven to be a useful and adaptable system for high-level expression ofcorrectly folded eukaryotic membrane proteins, thus they are an idealtool for the study of membrane-bound proteins. The large size of thecells, combined with the relatively short time needed for the expressionof the foreign plasma membrane-bound proteins (3-4 days) provides anexcellent experimental system for the application of isotope exchangetechniques. For expression in Sf9 insect cells, the Invitrogenbaculovirus Sf9 insect cell system is used. Expression vector constructs(pBluBac4.5, Invitrogen) encoding full-length AtNHX exchanger proteinsare prepared for each AtNHX and other PNHX isoforms using a PCR-basedsubcloning approach similar to that described above for the generationof GST-fusion proteins. Initially, the suitability of the insect cellexpression system for uptake analysis is performed using a single AtNHXisoform. The other PNHX isoforms are studied in a similar manner.Cultures of Sf9 insect cells are infected with baculovirus containingexpression vector constructs encoding the different PNHX isoforms.Infection and selection of transformants are performed as permanufacturer's instructions (Invitrogen). The isoform-specificantibodies described above aid in the assessment of recombinant proteinexpression and localization within the insect cells.

Equally important is the use of transgenic yeast as a tool for theexpression of recombinant eukaryotic proteins, particularly because ofpost-translational modifications and targeting to endomembranes. Inaddition, functional complementation of yeast mutant strains with plantproteins is often possible. We have subcloned the AtNHX1 cDNA into ayeast expression vector (pYES2) using a PCR-based approach as describedabove. Yeast (strain w303a) have been transformed with this constructand expression of the recombinant plant protein is confirmed once theantiserum is available. In addition, salt-tolerance of transformed yeastis assessed for each AtNHX isoform by comparing growth rates atdifferent NaCl concentrations. Methods for the isolation oftransport-competent plasma membranes and tonoplast and the isolation ofintact vacuoles are performed. The kinetics of H⁺/Na⁺ exchange ismeasured in intact insect cells and yeast, intact yeast vacuoles, andisolated plasma membranes and tonoplast vesicles according to knownmethods. Na⁺ influx in intact cells is monitored by isotopic exchangeusing [²²Na⁺]Cl and fast-filtration techniques [17,i,ii]. Kinetics ofH⁺-dependent Na⁺fluxes in vesicles is monitored by following thepH-dependent fluorescent quench of acridine dyes [13,17].

The results of these kinetic characterization studies providesinformation about the ion specificity, affinity, and optimal activityconditions for each AtNHX isoform. We assign the activity of eachisoform to the corresponding target membrane, and we also determinewhich of the isoforms have a higher affinity for sodium. We characterizethe mechanisms of salt tolerance in general and tissue-specificity anddevelopmental expression in particular.

In transgenic plants, expression of the different Na⁺/H⁺ antiports isverified with western blots using the isoform-specific antibodiesdescribed above. The kinetics of H⁺/Na⁺ exchange is measured in intactvacuoles, isolated plasma membranes and tonoplast vesicles (from rootsand leaves) as described above.

EXAMPLE 9

Identification of Positive and Negative Regulators of Na⁺/H⁺ AntiportActivity.

Heterologous expression of plant transport molecules in Saccharomycescerevisiae has been used successfully in recent years in numerousstudies. The availability of yeast mutants with salt-sensitivephenotypes (generated by ‘knock-outs’ of sodium transport molecules suchas Δenal-4- the plasma membrane Na⁺-ATPase pumps) makes it an especiallysuitable system for the study of sodium transport molecules. Thisheterologous expression facilitates kinetic studies of the antiportactivity in yeast cells using radiolabelled ²²Na⁺.

Successful suppression of yeast mutants, incapable of sodiumdetoxification allows for the genetic identification of positive andnegative regulators of these Na⁺/H⁺ antiports. Mutant yeast cells havinga suppressed phenotype as a result of the expression of a plant Na⁺/H⁺antiport are transformed with an Arabidopsis cDNA library for thepurpose of identifying particular regulators of these antiportmolecules. A phenotype of increased sodium tolerance in yeast identifiesparticular positive regulators of the antiport activity while negativeregulators are identified by a phenotype of decreased sodium tolerance.These phenotypes depend on the co-expression of the particular cDNAsidentified along with that of the Na⁺/H⁺ antiport under investigation.Identification of essential amino acid residues regulating the activityof Na⁺/H⁺ exchanger molecules is investigated by random mutagenesis ofthe antiport molecule which is achieved by PCR using a commerciallyavailable low fidelity Taq enzyme. The constructs generated are used intransforming sodium-related yeast mutants to identify particular Na⁺/H⁺antiport residues that affect suppression of the mutant yeast phenotype.Both gain-of-function and loss-of-function mutations are examined andmapped to the particular mutant residue by sequencing. Gain-of-functionmutations are of particular interest since they represent constitutiveactivation of the antiport activity allowing for increased sodiumdetoxification.

EXAMPLE 10

Transformation of Arabidopsis thaliana Using Overexpression of DifferentPutative Isoforms and Antiports From Other Plants, Preferably SaltTolerant Plants and Evaluation of Salt-tolerance

Arabidopsis represents a readily transformable model organism with theparticular advantage of having a short generation time. Agrobacteriumtumefaciens-mediated genetic transformation is utilized for Arabidopsis(ecotype Columbia). Studies include the overexpression of PNHXtransgenes in a wild-type background, combined overexpression of morethan one PNHX transgene, and suppression of endogenous PNHX expressionusing antisense PNHX expression. Stable transformation of progeny isconfirmed by Southern blotting. Overexpression of transgenes, orsuppression of expression using antisense constructs, is confirmed byNorthern and western blotting. In all cases, salt-tolerance oftransgenic plants is compared to wild-type plants, and-control plantstransformed with empty transformation vectors. Separate transformationsare performed on Arabidopsis plants using expression vector constructsfor each of the different AtNHX isoforms. In addition, Arabidopsisplants are transformed with PNHX genes from other plants, preferablysalt tolerant plants in order to assess the effect on salt tolerance ofthe expression of a Na⁺/H⁺ exchanger in a glycophytic plant.

For overexpression studies, full-length AtNHX2, AtNHX4, and AtNHX5 cDNAsare subcloned in a sense orientation into the expression vectorcontaining a “superpromoter” [20]. A PCR based subcloning strategy isused for each AtNHX cDNA as described above for the production ofNHXGST-fusion constructs. For the production of vector constructscontaining PNHX cDNAs in an antisense orientation, oligonucleotides withSalI and SacI restriction sites flanking the C-terminal and N-terminalPNHX regions respectively, are used for PCR amplification. All plasmidconstructs are sequenced on both strands to confirm the fidelity of thePCR amplification before transformation of Agrobaceterium tumefaciens(strain LBA4404). For each PNHX-pBISN1 construct, approximately 1 L ofAgrobaceterium culture, grown under antibiotic selection at 28° C, isused for the transformation of Arabidopsis. Plants are ready fortransformation when primary bolts are approximately 15 cm. About 2 flatsof plants (˜80 plants per flat) are used per transformation. A highlyefficient, vacuum-less infiltration transformation method [iii] is used.Harvested Agrobaceterium cultures are resuspended in an infiltrationmedia containing a mild surfactant (Silwet L-77, Lehle Seeds), and eachpot of Arabidopsis is simply submerged in the Agrobaceterium for 2-6minutes. Plants are thereafter drained, and returned to the growthchamber until the seeds are ready for harvesting (about 4 weeks). Seeds(T1 generation) are collected and after surface sterilization, areplated on sterile, selective media containing kanamycin, vernalized, andthen grown under optimal conditions. Healthy seedlings showing kanamycinresistence after about 7 days are transplanted to soil and the presenceof the transgene confirmed by Southern blotting. Seeds from T1transformants (ie T2 generation) are harvested, sown, and T2 plants usedfor Northern and western blotting to determine the expression patternsof the transgenes and PNHX proteins. Representative transgenic lines(e.g. showing low, medium, or high transgene expression) is used forstudies of salt-tolerance. A similar approach is used for transformationof Arabidopsis with the PNHXs from other plants.

Salt tolerance is assessed by measuring the growth rate of the plants atincreasing salt concentrations. Plant biomass, root/shoot ratios, tissueion content is measured. Root and hypocotyl growth rates is measured andcorrelated with tissue water content of plants growing at different NaClconcentrations.

EXAMPLE 11

Transformation of Crop Plants with A. thaliana and/or Other ExchangersUnder Constitutive and Inducible Promoters and Evaluation ofSalt-tolerance.

a) Agrobaceterium tumefaciens-mediated Transformation of Crop Plants

We assess whether or not homologues of the AtNHX genes exist in theplant of choice. We use degenerate oligonucleotide PCR-primers (asdescribed for other plants) and a cDNA library to isolate thefull-length cDNA. The high efficiency Agrobaceterium-mediatedtransformation method developed specifically for Brassica by Moloney etal [iv] is used to introduce and overexpress foreign nucleic acidmolecules and/or overexpress the endogenous PNHX nucleic acid moleculein the crop plant(s). This method lakes advantage of the fact that cutcotyledonary petioles from, which are capable of undergoingorganogenesis (ie generating explants), are very susceptible toAgrobaceterium infection. Shortly after germination (˜5 days) cotlyedonsare excised and imbedded into Murashige-Skoog medium (Gibco) enrichedwith benyzladenine. Expression vector constructs are prepared using aPCR-based subcloning approach as described above using the pCGN5059binary plasmid (which employs the CaMV 35S promoter to driveconstitutively high expression) engineered for gentamycin resistance[iv] and cDNAs of the various AtNHX clones and/or the halophyte PNHXclones, and the choosen plant PNHX clones. Excised cotyledons areinfected with Agrobaceterium cultures (strain EHA101), containing thevector construct of interest, by brief dipping and then co-cultivatedwith the Agrobaceterium for a 72 h. Subsequently, cotlyedons aretransferred to regeneration medium containing gentamycin as theselective agent. After explant regeneration, and subculturing, onselective media (˜4 weeks) explants are transferred to rooting mediumand then into soil once a root mass has developed. Tissue samples areexamined from growing plants to confirm transgene presence by Southernblotting as described above for the transformation of Arabidopsis.Transformed plants (T1 generation) are allowed to flower and set seedand these seeds are germinated (T2) under selective conditions andtransformants used for expression analysis of the transgenes andevaluation of salt-tolerance as described above. Also, biochemicalanalysis of the plants is performed. These include, Na⁺/H⁺ ratios,sugar, amino acid and quaternary N-compounds. Salt-tolerance is alsoevaluated in fields trials.

b) Microprojectile Bombardment-mediated Transformation of Crop Plants.

A microprojectile bombardment-mediated transformation of crop plants isused when Agrobacterium tumefaciens-mediated transformation is notsuccessful. We assess whether or not homologues of the AtNHX genes existin the plant of choice. We use degenerate oligonucleotide PCR-primers(as described above) and a cDNA library to isolate the full-length cDNA.Expression vector constructs, using the pBAR vector for high levelexpression of AtNHX or the halophyte PNHX or the endogenous PNHX fromthe plant of choice, are used in conjunction with the microprojectilebombardment system as described by Tomes et al. [v]. Bombardmentprocedures is carried out in callus tissue. Plant calli are initiated byculturing immature embryos on Callus medium [vi]. After about 2 weeks,friable calli that are growing rapidly are subcultured and grown for anadditional 2 weeks and then used for transformation. Calli fortransformation are transferred to fresh medium, incubated for 24 h andbombarded with tungsten microprojectiles carrying the pBARNHX vectorconstruct. Bombardment conditions is performed according tomanufacturer's instructions. Calli that show visible growth 10 daysafter bombardment are transferred to selective media (containing eitherBialaphos or Ignite) in order to identify putative transformants. Thegrowth of transformed plant calli on this selective media is continuedfor 3-4 months. Each putative stable transgenic event becomes apparentas a mass of friable embyogenic callus growing in the presence of theselection agent. Stable transformation is verified by Southern blots.Selected calli are transferred onto a regeneration medium [v], kept inthe dark at 28° C. for 7 days and then transferred to growth chambersunder a 16-h photoperiod until green shoots appear. Plantlets (1-2 cmlong) are transferred to individual tubes containing germination mediumto allow continued development. At the three to four leaf stage, plantsare transferred to soil and into the greenhouse. At the eight-leafstage, these plants are sprayed with 1% (w/v) Ignite herbicide to detectthe presence of the BAR gene. This herbicide kills those plants notcarrying the BAR gene. Confirmed transgenic plants (T1) are allowed tomature, flower, set seed, and seeds used for the production of T2plants. Transgenic T2 plants are used for the evaluation ofsalt-tolerance as described above. Transgenic T2 and T3 plants are usedin field trials for the evaluation of salt tolerance.

EXAMPLE 12

Cloning and Characterization of NX5

Material and Methods

Plant Material and Transformation:

Sterilized Arabidopsis thaliana seeds (Columbia) were grown either insoil directly or transferred to soil after germinating first on agarplates containing 0.5× Murashige and Skoog (MS) medium. The plants weregrown under 12 hour light at a constant 22° C.

Agrobacterium-mediated transformation was used to generate transgenicArabidopsis lines. Agrobacterium was resuspended in infiltrationsolution containing 0.5×MS salt, 0.5 g/l MES; 5% sucrose and 0.03%Silwet. Flowering plants with primary bolts reaching 15 cm were dunkedinto a bacterial solution for 5 min. The plants were re-transformed 12days later.

Transgenic seeds were screened on ½ MS-Kanamycin (25 mg/l) medium (½ MSK25). These plates were placed under 24 hour light at 22° C. The salttolerance of the transgenic lines was tested by either growing the seedson ½ MS-K25 plates—containing 100 mM NaCl and 200 mM NaCl or wateringthe plants in soil with 100 and 200 mM NaCl.

Arabidopsis seeds were sterilized as following: 50 μl of seeds werewashed with 1 ml 70% ethanol alcohol for 2 min, then incubated with 1 mlof sterilization solution with constant shaking for 10 min. Thesterilization solution contained 6% bleach and 0.1% Tween 20. Then theseeds were washed 5 times with sterilized ddH₂O. Finally, the seeds wereresuspended in 1 ml of 0.1% phytagar and chilled at 4° C. at leastovernight to break the dormancy.

Cloning of AtNHX5

5′- and 3′-Rapid Amplification of cDNA Ends were used to clone the fulllength of AtNHX5. SMART-RACE cDNA Amplification Kit and Advantage2 PCREnzyme System from CLONTECH were used. All reactions were performedaccording to the manufacturer instructions.

When compared with the conventional reverse transcription reactions, theRACE system provides a better mechanism for generating full-length cDNAsby using both the SMART II oligonucletide and MMLV reverse transcriptase(RT). This MMLV RT can add 3-5 residues of dC to the 3′ end of thefirst-strand cDNA, and these oligo-dC are harnessed by the dG-residuesof SMART II which serves as an extended primer for reversetranscription. Since the dc-tailing activity of RT is most efficientonly when the enzyme has reached the end of the RNA template, the SMARTII sequence is typically added only to complete first-strand cDNA, andthis guarantees the formation of cDNA that has a maximum amount of 5′sequence if high quality RNA is used. Furthermore, the Advantage 2polymerase Mix includes Advan Taq DNA polymerase, a minor amount of aproofreading polymerase, and Taq Start antibody to provide automatichot-start PCR, so this enzyme system allows efficient, accurate, andconventient amplification of cDNA.

5′-RACE to Obtain the 5′ End Start Codon of AtNHX5:

For the synthesis of first-strand cDNA (5′-RACE-ready cDNA), 1 μl (1.5μg) of total RNA, 1 μl of 5′-CDs primer (from the kit), 1 μl of SMART IIoligo (from the kit), and 2 μl of ddH₂O were mixed in a 0.5-ml PCR tube,and incubated at 70° C. for 2 min and placed on ice for 2 min. Then 2 μlof 5× first-strand synthesis buffer (from the kit), 1 μl of 20 mM DTT, 1μl of 10 mM dNTPs and 1 μl of Superscript II (MMLV RT) were added to thetube. Incubation was carried out at 42° C. for 1.5 hour. The reactionwas diluted by adding 100 μl of Tricine-EDTA buffer and the tube washeated at 72° C. for 7 min.

Tricine-EDTA buffer was used because this buffer can maintain pH at hightemperature better than Tris-EDTA buffer. Tris-based buffer can lead tolow pH conditions that can degree DNA.

For the 5′-RACE, an AtNHX5 specific 3′-primer, X5 REV, was designed,which is 5′-CCC CAA CCC CTG CAG ACA TTG AGC CAG C-3′ (SEQ ID NO: 8). ThePCR reaction was set up by combining the following: 5 μl of 10×advantage 2 PCR buffer, 1 μl of 10 mM dNTPs mix, 1 μl of 50× advantage 2polymerase mix, 5 μl of 5′-RACE-ready cDNA, 5 μl of 10× UPM, 1 μl ofX5REV, and 32 μl of PCR-grade ddH₂O. The PCR cycle included 1 cycle of94° C. for 2 min; 32 cycles of 94° C. for 30 sec and 68° C. for 3 min; 1cycle of 72° C. for 5 min. Negative control was done by adding no5′-RACE-ready cDNA.

3′-RACE to Obtain the Full-length of AtNHX5 Gene:

3′-RACE-ready cDNA was synthesized similarly to 5′-RACE-ready cDNA,except that 1 μl of 5′-CDs primer and SMART II oligo were replaced by3′-CDs primer attached with SMART II oligo.

For 3′-RACE, an AtNHX5 specific 5′ primer, X5-5′-RACE, was designed,which is 5′-GCT GAA TGG AGG AAG TGA TGA TTT CTC CGG TGG-3′ (SEQ ID NO:9). The 3′-RACE PCR reaction mixture included: 5 μl of 10× advantage 2polymerase buffer, 1 μl of 10 mM dNTPs, 1 μl of 50× advantage 2polymerase mix, 5 μl of 3′-RACE-ready cDNA, 1 μl of 5′-RACE primer, 5 μlof 10× UPM, and 32 μof PCR-grade ddH₂O. Negative controls were obtainedby adding no 3′-RACE-ready cDNA. PCR cycle included: 1 cycle of 94° C.for 2 min; 36 cycles of 94° C. for 30 sec and 72° C. for 3 min; 1 cycleof 72° C. for 7 min.

Preparation and Purification of Antibodies Raised Against the X5-GSTFusion Protein:

A specific region of 105 amino acids of ATNHX5 was chosen to make theX5-GST fusion protein.

First, the coding sequence of these 105 amino acids was obtained by PCRwith AtNHX5 cDNA and a pair of designed primers, GST-X5F (5′-CCC GCG GATCCG GTG CAC TTA TAT CAG C-3′ (SEQ ID NO: 10)) and GST-X5R (5′-GGC GGAATT CAC AAC ACT CCA AGT TCT G-3′ (SEQ ID NO: 11)). Then this piece ofDNA was cloned in frame into PGEX-2TK vector in the site of BamHI(5′-end) and E CoRI (3′-end).

E.Coli strain BL21 pLysS was used to express the fusion protein under 1mM IPTG induction. The fusion protein was confirmed by Western blotswith the anti-GST antibody. The E.Coli lysate was applied to apolyacrylamide gel, and then the fusion protein was cut off andsubjected to electroelution, lyophilization and concentrationmeasurement by dot blot.

For the 1^(st) injection of the rabbit, 200 μl (200 ug) of purifiedfusion protein was mixed by vortex with 200 μl Freund's completeadjuvant (ICN Biochemicals Inc.). For the 2^(nd), 3^(rd) and 4^(th) timeinjection, 250 μl (100-150 ug) of purified protein were mixed with equalvolume of Freund's incomplete adjuvant (ICN Biochemicals Ivc.).

For the purification of the antibody, affinity strip blots were used.For making one PVDF strip, 100 μg purified protein (GST or X5-GST fusionprotein) was loaded on a large well of a polyacrylaride gel. The stripswere blocked with 5% milk-PBST for 1 or 2 hours and washed with PBST for5 min three times before placed into the serum tube. The serum wasfirstly incubated with GST PVDF strips, and then with X5-GST fusionprotein stripes, at 4° C. overnight. The strips were washed with PBST 5times with 5 min each time, and then washed with 0.1× PBST three times.The antibody was eluted with 0.2 M Glycine freshly made, pH 2.5), andthe pH of the antibody solution was adjusted to 7.5 immediately byadding 2M Tris. Usually in 0.5 ml serum, 0.5 ml 2× PBS was added, and 2or 3 protein strips were used to bind the antibody. The purifiedantibody was stored at 4° C.

Yeast Complementation:

Yeast complementation was performed to test the function of AtNHX5.AtNHX5 ORF sequence was first obtained by PCR with AtNHX5 cDNA and apair of primers, 5′-X5-yeast-new (5′-CGC TCC CCC GGG ATG GAG GAA GTG ATGATT TCT CC-3′(SEQ ID NO: 12)) and 3′-X5-yeast-new (5′-GGA CGC GTC GACCTA CTC CCC ATC TCC ATC TCC-3′ (SEQ ID NO: 13)). The yeast mutant, Δnhx1(nutrition type:trp-), was transformed with AtNHX5 ORF sequence clonedin a yeast expression vector pYpGE15 at the SmaI (5′) and SalI (3′)sites. The wild type yeast and Δnhx1 mutant transformed with emptypYpGE15 vector, were also used as controls.

Transformation of Yeast:

The lithium acetate transformation method was used for thetransformation of yeast. Yeast mutant strain, Δnhx1, was streaked on atry-SD plate and incubated at 30° C. overnight. Several colonies werepicked and resuspended in 1 ml of sterile ddH₂O. The yeast cells werecentrifuged at 3500 rpm for 2 min. Yeast cells were washed with 300 μlof 100 mM LiAc, and finally resuspended in 150 μl of 100 mM LiAc (yeastcompetent cells). For transformation, 75 μl of the yeast competent cellswere mixed with 5 μl of plasmid, 5 μl of 10 μg/μl of denaturedsingle-stranded salmon sperm DNA and 300 μl of 40% PEG (in LiAc/TE), andthen incubated at 30° C. for 30 min without shaking. The yeast cellswere heat-shocked at 42° C. for 15 min to let the DNA go inside. Halfthe mixture was plated on a trp⁻-ura⁻-SD plate and the plate wasincubated at 30° C. for 2 days.

Salt Tolerance Test of the Transformants:

For testing the salt tolerance of the transformants, the yeast cellswere plated on APG medium with NaCl concentrations ranging from 200 to500 mM, and pH 4.5 and 5.5. As controls, wild type yeast Δnhx1 mutantand transformed with an empty pYpGE15 vector and plated on the samemedium plate with equal amount of cells. In order to plate equal amountof cell of every kind of yeast strains, all of the transformed yeaststrains were grown in 2 ml of trp⁻-ura ⁻-2% glucose-SD medium for 3 daysat 30° C., because at this point, all yeast cultures were saturated.Then dilutions of 10×, 100× and 1000× were made, and then 3 μl of eachculture were plated on APG plates containing 200 mM, 300 mM, 400 mM, and500 mM NaCl at pH 4.5 and pH 5.5. The plates were incubated at 30° C.

Semi-quantitative RT-PCR:

RT-PCR is a highly sensitive and rapid method of detecting mRNA levelsof a gene. It has been shown to be thousands of times more sensitivethan the traditional RNA blot techniques (Chirgwin et al., 1979;Chomczynski and Sacchi, 1987). Thus RT-PCR is more useful in analysis ofsingle-copy genes or lowly expressed genes. RT-PCR involves twoenzymatic reactions: a reverse transcription by usually MMLV RT, andpolymerase chain reaction. But it is difficult to get quantitativeinformation with this technique due to the exponential nature of PCR.Under ideal or theoretical conditions, the amount of product alwaysdoubles after each cycle, but the rate of production will reach aplateau stage after a certain number of cycles. There are severalfactors which may affect the amplification efficiency, such as theimpurity of the RNA sample, and the length of the amplified sequence.There may be additional factors that cause the tube-to-tube variationeven when a master mix of reaction components is used. So, in order toget accurate information, several strategies have been used in asemi-quantitative RT-PCR, including using an appropriate amount ofinitial template, appropriate numbers of PCR cycle and an endogenoussequence which is known to be expressed constantly as a internalcontrol. The internal control is amplified using a second pair ofspecific primers. Usually, a mini-southern blot is applied to detect thebands of the PCR products. The ratios of the amount of the targetproducts and that of the endogenous control represent the mRNA level ofa gene, and this is called normalization.

Usually, 2 μg of total RNA and 25 PCR cycle are used forsemi-quantitative RT-PCR. The RT-PCR beads from Amersham PharmaciaBiotech. Inc. was used for this thesis. To each RT-PCR bead (provided ina tube), 45 μl of DEPC-H₂O were added and dissolved on ice. Then 1 μl ofOligo dT (0.5 μg/μl), 0.5 μl of each of the four following primers,actin7F, actin7R, X5GSTF and X5-GSTR were added. Finally, 2 μl of 1μg/μl RNA sample was added. The totally volume of the PCR reaction was50 μl. The first cDNA strand synthesis was carried out at 42° C. for 30minutes. Then the tube was heated to 95° C. to inactivate the MMLV RT,then the normal PCR was started. The PCR condition were: 25 cycles of95° C. for 1 min, 55° C. for 1 min and 72° C. for 1 min. The sequence ofthe actin7F prime was 5′-GGT GAG GAT ATT CAG CCA CTT GTC TG-3′ (SEQ IDNO: 14), and that of the actin7R was 5′-TGT GAG ATC CCG ACC CGC AAGATC-3′ (SEQ ID NO: 15).

For detecting the RT-PCR product by mini-southern blot, the PCR productwas equally loaded in two agars gel wells and transferred to hybond-N⁺membrane by using 0.4 M NaOH overnight at room temperature.

For hybridization, one set of the membrane was probed with α-³²Plabelled actin 7 probe and the other set was probed with α-³²P labelledAtNHX5 specific probe. The RTPCR reactions were repeated several times,and all the results were subjected to statistical analysis.

Results

Cloning AtNHX5 cDNA

Through the screening of an Arabidopsis cDNA library, only partialsequence of AtNHX5 was obtained, which coded for 350 amino acids.According to this partial sequence, an AtNHX5 specific 3′-primer, X5 REVwas designed for 5′-RACE to obtain the 5′-end, and then an AtNHX5specific 5′-primer, X5-5′-RACE, was designed for 3′-RACE to obtain thefull length of AtNHX5. The open reading frame of AtNHX5 was a 1563-bpfragment (SEQ ID NO:4) which coded for 521 amino acids (SEQ ID NO:5)(FIG. 1). One amiloride binding site (FFLFLLPPII (SEQ ID NO: 16)) waslocated in the N-terminus of AtNHX5. The topology prediction (usingTopPred 2) showed that the full length AtNHX5 protein has 11transmembrane domains.

The Natural Subcellular Distribution of AtNHX5

In order to detect the subcellular location of AtNHX5, wild typeArabidopsis cellular membrane fractions, including tonoplast,mitochondria, plasma membrane, and ER, were prepared using sucrosegradient sedimentation, and 40 μg of each kind of membrane proteins wassubjected to Western-blotting by using antibody against AtNHX5. Theresult shows a strong band around 45 KDa exclusively located ontonoplast (FIG. 2, lane1). The molecular weight of the band was smallerthan expected, which was 56 Kda. This could be due to specific cleavageor modification of protein by the plant, or protein degradation, orabnormal migration caused by the interference of lipid component fromthe membrane.

The Expression Pattern of AtNHX5 and Its Response to Salt Treatment

30 μg of each kind of RNA sample was subjected to a formaldehydedenatured agarose gel electrophoresis and then transfered to Hybond-N+-membrane. The membrane was probed with α-³²P labeled AtNHX5 cDNA.Phosphore-image results showed a weak band around 2.1 kb in leaf, stemand root tissues, but not in the flower tissue, and there was no band inthe salt-treated leaf tissue either. In order to confirm theNorthern-blotting result, semi-quantitative RT-PCR was performed.Actin-7 was used as an internal control. The expected size of actin7band was 550 bp, and that of AtNHX5 was 315 bp. The semi-quantitativeRT-PCR results suggested that AtNHX5 was expressed in a slightly lowerlevel in flower than in the other tissues, but the difference was notstatistically significant.

To detect the expression of AtNHX5 indifferent developmental stages, RNAsample from 1-month old seedlings grown on MS-agar plates was preparedand subjected to semi-quantitative RT-PCR, and the results were comparedwith those results gotten from adult plant. No statistically significantdifference between the seedling sample and the adult samples at theAtNHX5 mRNA level was detected.

These results suggest that AtNHX5 might be expressed constantly fromyoung seedling to adult stage, and it does not response to salttreatment significantly.

The Function of AtNHX5

The function of AtNHX5 was tested by both yeast complementation andoverexpression in Arabidopsis.

Yeast Complementation

For yeast complementation experiment, the ORF of AtNHX5 was cloned intoa yeast expression vector, pYpGE 15, under PGK promoter, and theresulting construct (AtNHX5-pYpGE15) and the empty pYpGE15 vector wereused respectively to transform a yeast salt sensitive mutant, Δnhx1, byLithium actetate method (material and method). As a control, wild typeyeast strain was also transformed with empty pYpGE15 vector. The salttolerance of the different transformants was tested by plating equalamount of yeast cells of each kind on APG plates containing 200 mM NaClunder pH5.5. The result showed that the growth of Δnhx1 with emptypYpGE15 vector was inhibited by 200 mM NaCl, but AtNHX5 could suppressthe salt sensitivity of the Δnhx1 mutant strain (FIG. 2).

In order to check the effect of C-terminus of AtNHX5, a short version ofAtNHX5 ORF (called AtNHX5-S) which codes the first 350 amino acids wasalso used to do the yeast complementation experiment, and the resultshowed that AtNHX5-S could complement the mutated ScNHX1 function, butnot as well as the full ORF of AtNHX5 did. This result suggests that theC-terminus of AtNHX5 might have a regulatory function (FIG. 2).

Western-blotting was used to detect the expression in yeast of both thefull and short length of AtNHX5. Tonoplast samples from transformantswith full ORF and with short ORF of AtNHX5, as well as from Δnhx1 withempty pYpGE15 strain were prepared, and 60 μg of each sample was used todo Western blot. The results show a 57 KDa band for the full ORFtransformants (FIG. 2A), and a 40 KDa band for the short ORFtransformants (FIG. 2B). Both of these two bands had the expected sizes.

Based on the results described above, AtNHX5 may have the same functionas ScNHX1.

AtNHX5 Transgenic Arabidopsis Plants

The ORF of AtNHX5 was cloned into a plant expression vector (pBISN1)under the supermas promoter, with Kanamycin selection marker, at 5′ SalIsite and 3′ SmaI site. And Agrobacterium mediated transformationprotocol was used to obtain transgenic plants.

To transgenic seeds were first screened on MS-K25-agar plates. Totally15 lines survived. Then T₁ seeds of two lines (AtNHX5-L1 and AtNHX5-L2)were plated on MS-K25-agar plates containing 50, 100, 150, and 200 mMNaCl. There were several seedlings from each of the two lines, whichsurvived on 50 and 100 mM NaCl plates, but not on the 150 mM NaCl andabove plates. Then the surviving seedlings from the 100 mM NaClMS-K25-agar plates were transfered to soil. Ten days after the transfer,a set of three plants of each line were started to be watered with 0,100, and 200 mM NaCl solutions every other day. Meantime, as controls, aset of wild type plants were also treated the same way. After wateredwith 200 mM NaCl for 2 weeks, the wild type plant was almost deadcompletely, while one transgenic line, AtNHX5-L2, was still as green asits control plant which was watered with 0 mM NaCl.

The mRNA level of AtNHX5 in both AtNHX5-L2 line and wild type weretested. RNA samples from young seedlings of AtNHX5-L2 line and wild type(from MS plates) were prepared and subjected to semi-quantitativeRT-PCR. The same AtNHX5 specific primers and actin7 control primers usedfor detecting the tissue distribution of AtNHX5 were used here. Theresults showed that AtNHX5 mRNA was about 4 times higher in AtNHX5-L2line than in wild type. Therefore, overexpression of AtNHX5 inArabidopsis can confer a higher salt tolerance to the plants.

EXAMPLE 13

Cloning and Characterization of NX4

PCR primers were designed for the amplification of the AtNHX2 sequencebased on a BAC DNA sequence (T9J14.2) with a predicted amino acidsequence that showed homology to AtNHX1:

-   -   X2F, 5′-TTCGCCTCTTTAACCTCTAAAATG-3′ (SEQ ID NO: 17)    -   X2R, 5′-TGTAGGCAAGAGCCATAGATACAG-3′ (SEQ ID NO: 18)

A single band of approximately 1.2 kb was amplified from a sizefractionated flower cDNA library (CD4-6, ABRC) and was used as atemplate for screening the flower cDNA library with ³²P-labeled probes.Plaques hybridizing to the probe in the first round were subjected to asecond round using the same probe. The largest clone obtained fromexcised phagemids from the second round of screening was incomplete atthe N-terminus coding portion of the gene. The 5′ end of the cDNA wasfirst determined using 5′ -rapid amplification of cDNA ends (RACE). A5′-RACE primer, which primes transcription towards the 5′-end of thecDNA, was designed for use with the SMART RACE cDNA amplification kit(Clontech):

-   -   X2-5′-RACE1,5′-TACAGAGTCGGTTGCAGCAAATATGGCG-3′ (SEQ ID NO: 19)

5′-RACE was performed with 3 μg of RNA isolated from flower tissues ofA.thaliana (Col) according to manufacturer's instructions. A single 800bp fragment was amplified, cloned into Invitrogen pCR2.1 TOPO vectorusing the Invitrogen TOPO TA Cloning Kit according to the manufacturer'sinstructions. Using the most extreme 5′-sequence from the 5′-RACE andthe most extreme 3′-sequence from the original screen, two primers weredesigned for amplification of the full-length cDNA:

-   -   X2-3′END, 5′-CACCAATACTAGTCACCATAAGAGGGAAGAGCA-3′ (SEQ ID NO:        20);    -   X2-5′END, 5′-CTGCCTCTCTCTCAACGCAACTCAATCCA-3′ (SEQ ID NO: 21)

Using these primers and the RACE-ready cDNA prepared from RNA isolatedfrom flower tissues of A.thaliana (Col), a 2.1 kb product was amplifiedaccording to manufacturer's instructions. This was submitted forcomplete sequencing and compared with sequences obtained from theoriginal library clone and RACE product. The full length nucleotidesequence (SEQ ID NO:1) and the derived protein sequence (SEQ ID NO:2)are shown in FIG. 1.

EXAMPLE 14

Cloning and Characterization of NX4

PCR primers were designed for the amplification of the AtNHX2 sequencebased on a BAC DNA sequence (F24P17.16) with a predicted amino acidsequence that showed homology to AtNHX1. The cloning and sequencing ofAtNHX5 was undertaken by Dr. Xue-Jun Hua. The full-length AtNHX4 cDNAwas isolated from the Δ-PRL-2 Arabidopsis cDNA library (ABRC).

RT-PCR

Unique regions of each cDNA were selected for amplification of probetemplates so that we could be certain that signals, when detected forthe in situ hybridization as with the RT-PCR above, were specific forthe particular gene of interest. The following primer pairs, given hereas gene name, region of the cDNA from which it was amplified, ampliconsize, and then primer pair, were used to amplify PCR products forcloning into the pSPT18 and pSPT19 cloning vectors (Roche, Indianapolis,Ind.):

AtNHX2, 5′-UTR, 353 bp

-   -   X2ECO 5′-GAATTCCTCAACGCAACTCAATCCAC-3′ (SEQ ID NO: 22)    -   X2PST 5′-CTGCAGGGCGAACATTGTCATCTTTC-3′ (SEQ ID NO: 23)        AtNHX4, 3′-UTR, 474 bp    -   X4ECO 5′-GAATTCCATTGAGAATAGTGTTCCGCAA-3′ (SEQ ID NO: 24)    -   X4PST 5′-CTGCAGGATTCGTGTCCCTTTGTTTTG-3′ (SEQ ID NO: 25)        AtNHX5, central portion of ORF, 502 bp    -   X5ECO 5′-GAATTCTCGCTTCAGTTGTFACTGGTG-3′ (SEQ ID NO: 26)    -   X5PST 5′-CTGCAGCGCTTCATAACAATTCCTGTCA-3′ (SEQ ID NO: 27)

DIG-labeled sense and antisense probes were synthesized and labelingefficiency was evaluated according to manufacturer's instructions (DIGRNA Labeling Kit, Roche, Indianapolis, Ind.).

In Situ Hybridization

Unique regions of each cDNA were selected for amplification of probetemplates so that we could be certain that signals, when detected forthe in situ hybridization as with the RT-PCR above, were specific forthe particular gene of interest. The following primer pairs, given hereas gene name, region of the cDNA from which it was amplified, ampliconsize, and then primer pair, were used to amplify PCR products forcloning into the pSPT18 and pSPT19 cloning vectors (Roche, Indianapolis,Ind.):

AtNHX2, 5′-UTR, 353 bp

-   -   X2ECO 5′-GAATTCCTCAACGCAACTCAATCCAC-3′    -   X2PST 5′-CTGCAGGGCGAACATTGTCATCTTTC-3′        AtNHX4, 3′-UTR, 474 bp    -   X4ECO 5′-GAATTCCATTGAGAATAGTGTTCCGCAA-3′    -   X4PST 5′-CTGCAGGATTCGTGTCCCTTTGTTTTG-3′        AtNHX5, central portion of ORF, 502 bp    -   X5ECO 5′-GAATTCTCGCTTCAGTTGTTACTGGTG-3′    -   X5PST 5′-CTGCAGCGCTTCATAACAATTCCTGTCA-3′        DIG-labeled sense and antisense probes were synthesized and        labeling efficiency was evaluated according to manufacturer's        instructions (DIG RNA Labeling Kit, Roche, Indianapolis, Ind.).

In situ hybridization was performed as described by Long et al. (1996),except for the following modifications. Arabidopsis thaliana ecotypeColumbia plant tissues were fixed in 4% paraformaldehyde and dehydratedthrough an ethanol and ter-butyl alcohol series before embedding inparaffin. Seven-μm-thick sections were adhered to ProbeOn Plus slides(Fisher, Pittsburgh, Pa.), deparaffinized with Histoclear, rehydratedthrough an ethanol series, treated for 30 min with 1 μg/mL proteinase K,refixed with paraformaldehyde, treated with 0.1 M acetic anhydride, anddehydrated through an ethanol series. Slide pairs were incubated withequal concentrations of sense and antisense probes at 55° C. overnight.Post-hybridization treatment, including washes and RNAse treatment, wasalso as described by Long et al. (1996). Alkaline-phosphatase conjugatedanti-DIG antibodies were used to detect DIG-labeled probes. NBT/BCIP(Invitrogen, Carlsbad, Calif.) was incubated with the slides to generatea color reaction with the alkaline phosphatase bound to the antibody.Slides were dehydrated and mounted with Permount (Sigma, St. Louis,Mo.).

In situ hybridizations for detection of AtNHX2 and AtNHX4 echo theresults of the RT-PCR. AtNHX2 shows specific floral expressionparticularly in the septum of the carpel. Signals for antisense probesof AtNHX2 were not detected in other tissues. AtNHX4 signal was observedonly in roots. AtNHX2, although detectable in tapetal cells, is moststrongly expressed in the septum of the developing silique. AtNHX4showed a very tissue-specific expression, both by RT-PCR and in situanalyses. Signal for AtNHX4 expression was detectable only in roots;however, no cell-specific conclusions can be made regarding AtNHX4expression because of the poor preservation of root sections in the insitu protocol.

The present invention has been described in detail and with particularreference to the preferred embodiments; however, it will be understoodby one having ordinary skill in the art that changes can be made theretowithout departing from the spirit and scope of the invention.

All articles, patents and other documents described in this application(including Genbank sequences and/or accession numbers), U.S. applicationNo. 60/078,474 (filed Mar. 18, 1998), U.S. application No. 60/116,111(filed Jan. 15, 1998) and U.S. Pat. Nos. 5,612,191, 5,763,211, 5,750,848and 5681714, are incorporated by reference in their entirety to the sameextent as if each individual publication, patent or document wasspecifically and individually indicated to be incorporated by referencein its entirety. They are also incorporated to the extent that theysupplement, explain, provide a background for, or teach methodology,techniques and/or compositions employed herein.

The following is a non-limiting list of plants which find use in thepresent invention: Alfalfa, Almond, Apple, Apricot, Arabidopsis,Artichoke, Atriplex, Avocado, Barley, Beet, Birch, Brassica, Cabbage,Cacao, Cantaloup/cantalope, Carnations, Castorbean, Caulifower, Celery,Clover, Coffee, Corn, Cotton, Cucumber, Garlic, Grape, Grapefruit, Hemp,Hops, Lettuce, Maple, Melon, Mustard, Oak, Oat, Olive, Onion, Orange,Pea, Peach, Pear, Pepper, Pine, Plum, Poplar, Potato, Prune, Radish,Rape, Rice, Roses, Rye, Sorghum, Soybean, Spinach, Squash, Strawberries,Sunflower, Sweet corn, Tobacco, Tomato and Wheat

REFERENCES

-   [1] Rush, P W and Epstein, E (1981). J. Amer. Soc. Hort. Sci. 106,    699-704.-   [2] Norlyn, J D (1980). In: Genetic Engineering of Osmoregulation    (Eds. D W Rains, R C Valentine and A Hollaender) pp. 293-309. Plenum    Press: New York.-   [3] Tal, M (1985). Plant & Soil 89, 199-226.-   [4] Flowers, T J and Yeo, A R (1995). Aust. J. Plant Physiol. 22,    875-884.-   [5] Bonhert, H J and Jensen, R G (1996). Aust J. Plant Physiol. 23,    661-667.-   [6] Tarcynski, M C, Jensen, R G & Bonhert, H J. (1995) Science 259,    508-510.-   [7] Kishor et al. (1 995). Plant Physiol. 108, 1387-1394.-   [8] Ishitani, M, et al., (1995). Plant Mol. Biol. 27, 307-317-   [9] Xu, et al. (1996) Plant Physiol. 110, 249-257.-   [10] Wu, R and Ho, THD. Patent # W09713843.-   [11] Jia, Z P, et al., (1992). EMBO J. 11, 1631-1640.-   [12] Young, P G & Zheng, P. J. Patent #W09106651.-   [13] Blumwald, E & Poole, R. J. (1985) Plant Physiol. 78, 163-167.-   [14] Blumwald, E et al., (1987). Plant Physiol. 85, 30-33.-   [15] Blumwald, E & Poole, R. J. (1987) Plant Physiol. 83, 884-887.-   [16] Barkla, B J, et al., (1990). Plant Physiol. 93, 924-930.-   [17] Barkla, B. J. & Blumwald, E. (1991) Proc. Natl. Acad. Sci. USA    88, 11777-11181.-   [18] Blumwald, E. & Gelli, A. (1997). Adv. Bot. Res. 25, 401-417.-   [19] Thompson, J D et al., Nucleic Acid Res. 22:4673-4680.-   [20] Ni et al., (1995) Plant Journal 7:661-676-   [21] Shah et al., (1986) Science 233:478-481.-   [22] Ono et al., (1996) Plant Physiol 112:483-491-   [23] Abe et al., (1997) Plant Cell 9:1859-1868.-   [24] Rieping M and Schoffl F (1992) Mol Gen Genet 231:226-232].-   [25] Raghothama et al., (1997) Plant Mol Biol 34:393-402].-   [26] Mett et al., (1996) Transgenic Res 5:105-113.-   [27] Schena et al., (1991) PNAS 88:10421-10425.-   [28] Vorst et al. (1990) Plant Mol Biol 14:491499.-   [29] Wanapu & Shinmyo (1996) Ann. NY Acad. Sci. 782:107-114.-   [30] Harlow E & Lane D (1988). Antibodies: a laboratory manual. Cold    Spring Harbor Laboratory Press. New York.-   [31] Sambrook, J, Fritsch, E. E. & Maniatis, T. (1989). Molecular    Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press.    New York.-   [32] Lee, C. C. & Caskey, T. in: PCR Protocols: A guide to Methods    and Applications. Academic Press, Inc. San Diego. pp.46-53-   [33] Narasimhulu, S B, et al., Plant Cell 8:873-886 [1996])-   [34] McCormac A C. et al., (1997) Mol Biotechnol. 8:199-213.-   [35] Ma H, et al., (1987) Gene 58:202-226-   [36] Gietz R D & Woods, R A (1994). High efficiency transformation    with lithium acetate. In Molecular Genetics of Yeast, A Practical    Approach (J. Johnston, ed.) New York: IRL Press. pp.121-134.-   [37] Gietz, R D & Sugino, A (1988), Gene 74: 527-534.-   [38] Krieber et al. (1993) Cell 72:427441.-   [i] Blumwald, E. (1987). Physiol. Plant 69, 731-734.-   [ii] Blumwald, E. & Poole, R. J. (1986). Plant Physiol. 80,727-731.-   [iii] Clough, M. & Bent, A. (1997). Arabidopsis Meeting, Madison,    Wis.,-   [iv] Moloney, M. M., Walker, J. M. & Sharma, K. K. (1989). Plant    Cell Rep. 8, 238-242.-   [v] Tomes, D. T. C., Ross, M. C., & Songstad, D. D. (1995). In:Plant    Cell, Tissue and Organ Culture (O. L. Gamborg & G. C. Phillips,    eds). Springer, New York. pp 197-213.-   [vi] Amstrong, C. L., & Green, C. E. (1985). Planta 164,207-214.

1. An isolated nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:5.
 2. A vector comprising the nucleic acid molecule ofclaim
 1. 3. The vector of claim 2, wherein a promoter is operativelylinked to the nucleic acid molecule, wherein the promoter is selectedfrom the group consisting of a super promoter, a 35S promoter ofcauliflower mosaic virus, a drought-inducible promoter, an ABA-inducible promoter, a heat shock-inducible promoter, a salt-induciblepromoter, a copper-inducible promoter, a steroid-inducible promoter anda tissue-specific promoter.
 4. A host cell comprising vector of claim 2.5. The host cell of claim 4, wherein said host cell is selected from thegroup consisting of a fungal cell, a yeast cell, a bacterial cell, abacterial cell and a plant cell.
 6. A transgenic plant, a plant part, aseed, a plant cell or progeny thereof, wherein the plant, plant part,seed, plant cell or progeny comprises the nucleic acid molecule ofclaim
 1. 7. The plant part of claim 6, comprising all or part of a leaf,a flower, a stem, a root or a tuber.
 8. The plant, plant part, seed orplant cell of claim 6, wherein the plant, plant part, seed or plant cellis of a species selected from the group consisting of potato, tomato,brassica, cotton, sunflower, strawberries, spinach, lettuce, rice,soybean, corn, wheat, rye, barley, atriplex, sorgum, alfalfa, salicorniaalmond, apple, apricot, arabidopsis, artichoke, avocado, beef, birch,cabbage, cacao, cantaloupe/cantaloup, carnations, castorbean,cauliflower, celery, clover, coffee, cucumber, garlic, grape,grapefruit, hemp, hops, maple, melon, mustard, oak, oat, olive, onion,orange, pea, peach, pear, peanuts, pepper, pine, plum, poplar, prune,radish, rape, rose, squash, sweet corn, and tobacco.
 9. The plant, plantpart, seed or plant cell of claim 6, wherein the plant is a dicot plant.10. The plant, plant part, seed or plant cell of claim 6, wherein theplant is a monocot plant.
 11. A transgenic plant comprising recombinantDNA, wherein the recombinant DNA encodes a polypeptide comprising thepeptide sequence of SEQ ID NO:6.
 12. A transgenic plant comprising arecombinant nucleic acid molecule encoding a polypeptide having Na⁺/H⁺transporter activity that provides increased salt tolerance in a cell,wherein said nucleic acid molecule is selected from the group consistingof: (a) the isolated nucleic acid molecule of claim 1, (b) a nucleicacid molecule encoding SEQ ID NO:6; and (c) a nucleic acid moleculeencoding a protein that is at least 95% identical to SEQ ID NO:6. 13.The transgenic plant of claim 12, wherein the polypeptide comprise anAtNHX transporter polypeptide having Na+/H+ transporter activity thatprovides increased salt tolerance in a cell.
 14. The transgenic plant ofclaim 12, wherein the recombinant nucleic acid molecule is operativelylinked to a constitutive promoter sequence or an inducible promotersequence, wherein the promoter provides transcription of the recombinantnucleic acid molecule in the plant.
 15. The transgenic plant of claim12, wherein the recombinant nucleic acid molecule is chemicallysynthesized.
 16. The transgenic plant of claim 12, wherein therecombinant nucleic acid molecule is isolated from Arabidopsis thaliana.17. The transgenic plant of claim 12, wherein the polypeptide havingNa+/H+ transporter activity extrudes monovalent cations out of thecytosol of a first cell of said plant transformed with said recombinantnucleic acid molecule to provide the first cell with increased salttolerance relative to a second non-transformed cell of a plant of thesame variety as said transgenic plant, wherein the monovalent cationsare selected from at least one of the group consisting of sodium,lithium and potassium.
 18. The transgenic plant of claim 17, wherein themonovalent cations are extruded into a vacuole or into the extracellularspace of said plant.
 19. An expression transgene comprising arecombinant nucleic acid molecule operably linked to a promoter selectedfrom the group consisting of a super promoter, a 355 promoter ofcauliflower mosaic virus, a drought-inducible promoter, an ABA-induciblepromoter, a heat shock-inducible promoter, a salt-inducible promoter, acopper-inducible promoter, a steroid-inducible promoter and atissue-specific promoter, wherein said nucleic acid molecule is selectedfrom the group consisting of: (a) the isolated nucleic acid molecule ofclaim 1, (b) a nucleic acid molecule encoding SEQ ID NO:6, and (c) anucleic acid molecule encoding a protein that is at least 95% identicalto SEQ ID NO:6.
 20. A plant cell or progeny thereof, comprising theexpression transgene of claim
 19. 21. A plant, a plant part, a seed, aplant cell or progeny thereof, wherein the plant, plant part, seed,plant cell, or progeny thereof comprise the expression transgene ofclaim
 11. 22. The plant part of claim 21, comprising all or part of aleaf, a flower, a stem, a root or a tuber.
 23. The plant, plant part,seed or plant cell of claim 21, wherein the plant, plant part, seed orplant cell is of a species selected from the consisting of potato,tomato, brassica, cotton, sunflower, strawberry, spinach, lettuce, rice,soybean, corn, wheat, rye, barley, atriplex, sorghum, alfalfa,salicornia, almond, apple, apricot, arabidopsis, artichoke, avocado,beet, birch, cabbage, cacao, cantaloupe/cantaloup, carnations,castorbean, cauliflower, celery, clover, coffee, cucumber, garlic,grape, grapefruit, hemp, hops, maple, melon, mustard, oak, oat, olive,onion, orange, pea, peach, pear, peanuts, pepper, pine, plum, poplar,prune, radish, rape, rose, squash, sweet corn, and tobacco.
 24. Theplant, plant part, seed or plant cell of claim 21, wherein the plant isa dicot plant.
 25. The plant, plant part, seed or plant cell of claim21, wherein the plant is a monocot plant.
 26. A method for producing arecombinant plant cell that expresses a nucleic acid molecule, whereinthe method comprises introducing into the plant cell the expressiontransgene of claim
 19. 27. A method of producing a geneticallytransformed plant which expresses PNHX transporter polypeptide whereinthe method comprises regenerating a genetically transformed plant fromthe plant cell, seed or plant part of claim
 21. 28. The method of claim26, wherein the genome of the plant cell also comprises a functionalPNHX gene.
 29. The method of claim 26, wherein the genome of the plantcell does not comprise a functional PNHX gene.
 30. A transgenic plantaccording to the method of claim
 27. 31. A method for expressing a PHNXtransporter polypeptide in a plant cell, wherein the method comprisesculturing the plant cell of claim 20 under conditions suitable for geneexpression.
 32. A method for producing a transgenic plant that expresseselevated levels of PNHX transporter polypeptide relative to anon-transgenic plant, comprising transforming a plant with theexpression transgene of claim
 19. 33. A method of producing agenetically transformed plant, wherein the method comprises: (a) cloningor synthesizing a nucleic acid molecule selected from the groupconsisting of: (i) the isolated nucleic acid molecule of claim 1, (ii)nucleic acid molecule encoding SEQ ID NO:6, (iii) a nucleic acidmolecule encoding a protein that is at least 95% identical to SEQ IDNO:6; (b) inserting the nucleic acid molecule in a vector so that thenucleic acid molecule is operably linked to a promoter; (c) insertingthe vector into a plant cell or plant seed; and (d) regenerating a plantfrom the plant cell or plant seed, wherein salt tolerance in the plantis increased compared to a wild type plant.
 34. A transgenic plantproduced according to the method of claim 33.